marine drugs

Review

Sea Cucumber Derived Type I Collagen:A Comprehensive Review

Tharindu R.L. Senadheera 1 , Deepika Dave 1,2,* and Fereidoon Shahidi 1,*1 Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL A1B 3X9, Canada;

trlsenadheer@mun.ca2 Marine Bioprocessing Facility, Centre of Aquaculture and Seafood Development, Fisheries and Marine

Institute, Memorial University of Newfoundland, St. John’s, NL A1C 5R3, Canada* Correspondence: Deepika.Dave@mi.mun.ca (D.D.); fshahidi@mun.ca (F.S.)

Received: 28 August 2020; Accepted: 15 September 2020; Published: 18 September 2020�����������������

Abstract: Collagen is the major fibrillar protein in most living organisms. Among the different typesof collagen, type I collagen is the most abundant one in tissues of marine invertebrates. Due tothe health-related risk factors and religious constraints, use of mammalian derived collagen hasbeen limited. This triggers the search for alternative sources of collagen for both food and non-foodapplications. In this regard, numerous studies have been conducted on maximizing the utilization ofseafood processing by-products and address the need for collagen. However, less attention has beengiven to marine invertebrates and their by-products. The present review has focused on identifyingsea cucumber as a potential source of collagen and discusses the general scope of collagen extraction,isolation, characterization, and physicochemical properties along with opportunities and challengesfor utilizing marine-derived collagen.

Keywords: sea cucumber; collagen; characterization; physicochemical properties; applications

1. Introduction

During the past few decades, sea cucumbers and other marine invertebrates have been exploitedfor commercial use for food and health purposes. Recently, seafood and its derivatives have becomeone of the most traded food commodities around the globe for disease risk reduction and healthpromotion [1–3]. The trend further enhanced the popularity of this marine species among the scientificcommunity as well as consumers [4,5]. Apart from the high nutritional profile of sea cucumber, theavailability of unique bioactive compounds coupled with the therapeutic properties has upgraded theirposition as a functional food ingredient. Bioactive compounds including antioxidant, antihypertensive,anti-inflammatory, anticancer, antimicrobial, and anticoagulant/antithrombotic compounds have beenidentified in different sea cucumber species available around the world [2,6]. Moreover, sea cucumbersplay a vital role as echinoderms in the marine ecosystems and are primary organisms comprised ofgrazing, predation and bioturbation in benthic areas and deep oceans across the world [7]. Hence,it becomes essential to utilize these marine resources sustainably with consideration of environmentaland commercial perspectives.

The versatile nature of the unique functional and technological properties of sea cucumberhas extensively been studied and has shown great potential for developing novel foods as well asbio-medicinal applications [8–10]. The groups of bioactive compounds identified with elucidatedstructures are collagen, gelatin, saponins, chondroitin sulphates, glycolipids, triterpene glycosides,mucopolysaccharides, bioactive peptides, vitamins, minerals, carotenoids, and amino acids, amongothers [5,11–13]. Within these numerous bioactive compounds, sea cucumber has been considered asa rich source of collagen [14]. As soft-bodied marine invertebrates, sea cucumbers possess leathery

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skin and an elongated body with the body wall attaining the highest market demand [15]. Recently,sea cucumbers have gained increased attention as one of the primary sources for high-quality marinecollagen as alternatives to mammalian collagen due to the recent occurrences of pathogenic diseasesand religious sentiments [8,16,17]. However, the amount and the nature of collagen available insea cucumbers depend on the species, biological environment, and diet. The most recent study onCucumaria frondosa demonstrated that extractable collagen from its body wall is less than a fraction ofone percent [18]. This could be related to the feeding habit of Cucumaria frondosa that is associated withthe phytoplankton, zooplankton, and other organic matter whereas other species feed on mud anddead particles on the sea floor.

Considering the collagen as a biomaterial, it has many usages in several fields [19]. Application ofcollagen is diversified mainly due to its unique properties such as biocompatibility, low antigenicity,high biodegradability, and cell growth potential [17]. Apart from the food industry, collagens havebeen widely used in tissue engineering, pharmaceutical, and biomedical industries as well as variousother fields, including cosmetics [20]. For these applications, the quality and purity of collagen play asignificant role. Besides the origin of raw material, extraction conditions might have a direct influenceon the yield and properties of the resultant collagen. The extracted crude collagen requires downstreamprocessing for purification based on the quality of crude collagen and is subsequently available for awide range of applications covering both medical and industrial domains.

Furthermore, to obtain high-quality collagen, it is crucial to consider pre-treatment steps priorto extraction procedures. In addition, paying attention to the recovery of collagen is beneficial, bothfrom the isolation and purification methodology standpoints [10]. In addition, characterization of theextracted collagen is another key factor that helps identifying its potential as a biomaterial in diversifiedapplications. Therefore, exploring novel approaches to extract the collagen from sea cucumber with lessenvironmental impact and studying their application areas have attracted the interest of researchersfrom both scientific and industrial communities. The present review provides some backgroundinformation about collagen and explains its different sources including that from sea cucumber withemphasis on the extraction, isolation, purification, and characterization techniques mainly related totype I collagen. In addition, use of collagen and its functional properties as well as challenges andfuture perspectives of utilizing collagen from sea cucumbers are discussed.

2. Definition and History of Collagen

As one of the most abundant fibrous proteins, collagen plays a vital role in connective tissues, thusanimal skin and bone provide an extracellular framework for strength and flexibility [9]. Collagen isone of the major structural proteins in the extracellular matrix and the name is derived from theGreek word “kola,” which means “glue producing”. Moreover, “collagen” is considered a genericterm, and no well-defined criteria exist to name this structural biopolymer [21,22]. Findings ofSchweitzer et al. [23] revealed the presence of intact collagen in the soft tissue of the fossilized bones of68 million-year-old Tyrannoaurus rex, a genus of coelurosaurian theropod dinosaur. Sequences of studieshave been conducted for decades to propose a structure for the collagen molecule. Among thosestudies, triple-helical “Madras Model” by Ramachandran and Kartha [24] contributed much to thecurrently accepted structure of collagen which was discovered by Cowan, North and Randall [25].Further findings of Rich and Crick [26] also improved the identified structure of collagen [27]. Currently,more than 29 distinct types of collagen have been identified [28,29].

The molecular structure of collagen consists of three polypeptide α chains intertwined with eachother to form a triple helix, approximately 300 nm in length with a molecular weight of 105 kDa [8].These molecules can either be homomeric (contain identical α chains) or heteromeric (geneticallydistinct α chains) [30]. Each strand is initially shaped into a left-handed symmetry prior to theirconformation as a right-handed triple helix.

Each chain of the right-handed helical structure consists of a repeating sequence of glycine-X-Y,where often, X and Y are referred to proline or hydroxyproline, respectively (Figure 1) [29]. In this

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motif, all glycine residues are located inside the core, while other amino acids (X and Y) are located onthe surface [30]. This rigid rod-like structure is further strengthened by interchain N-H (Gly) O=C(x)hydrogen bonds and electrostatic interactions [31]. The presence of triple helix (Figure 2) is the mainfeature in the collagen structure. However, triple helix can be varied according to the type of collagenpresent in the structure [32]. Moreover, this sequential uniformity can rarely be found in other proteins.Due to the uniformity of collagen, numerous studies have been conducted to determine their potentialas a prospective biomaterial for a wide range of applications.

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O=C(x) hydrogen bonds and electrostatic interactions [31]. The presence of triple helix (Figure 2) is the main feature in the collagen structure. However, triple helix can be varied according to the type of collagen present in the structure [32]. Moreover, this sequential uniformity can rarely be found in other proteins. Due to the uniformity of collagen, numerous studies have been conducted to determine their potential as a prospective biomaterial for a wide range of applications.

Figure 1. Amino acid residues present in triple helix.

Figure 2. Triple helix structure of collagen.

2.1. Basic Structure and Synthesis

The fundamental subunit of collagen is tropocollagen which is a three-stranded polypeptide unit. Collagen family is classified into various groups due to their complex structural diversity [21]. Different lengths of the helix, presence of non-helical components, interruptions in the helix, variations in the assembly of the basic polypeptide chains, and differences in the terminations of the helical domain directly lead to distinct types of collagens. Its general groups include fibrillar collagens, FACIT (Fibril Associated Collagens with Interrupted Triple Helices), FACIT-like collagen, beaded filament collagen, basement membrane collagen, short-chain collagen, transmembrane collagen, and unclassified collagen [29,33]. The length of the helix and portions of non-helical components are different depending on the type of collagen.

Figure 1. Amino acid residues present in triple helix.

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O=C(x) hydrogen bonds and electrostatic interactions [31]. The presence of triple helix (Figure 2) is the main feature in the collagen structure. However, triple helix can be varied according to the type of collagen present in the structure [32]. Moreover, this sequential uniformity can rarely be found in other proteins. Due to the uniformity of collagen, numerous studies have been conducted to determine their potential as a prospective biomaterial for a wide range of applications.

Figure 1. Amino acid residues present in triple helix.

Figure 2. Triple helix structure of collagen.

2.1. Basic Structure and Synthesis

The fundamental subunit of collagen is tropocollagen which is a three-stranded polypeptide unit. Collagen family is classified into various groups due to their complex structural diversity [21]. Different lengths of the helix, presence of non-helical components, interruptions in the helix, variations in the assembly of the basic polypeptide chains, and differences in the terminations of the helical domain directly lead to distinct types of collagens. Its general groups include fibrillar collagens, FACIT (Fibril Associated Collagens with Interrupted Triple Helices), FACIT-like collagen, beaded filament collagen, basement membrane collagen, short-chain collagen, transmembrane collagen, and unclassified collagen [29,33]. The length of the helix and portions of non-helical components are different depending on the type of collagen.

Figure 2. Triple helix structure of collagen.

2.1. Basic Structure and Synthesis

The fundamental subunit of collagen is tropocollagen which is a three-stranded polypeptideunit. Collagen family is classified into various groups due to their complex structural diversity [21].Different lengths of the helix, presence of non-helical components, interruptions in the helix, variationsin the assembly of the basic polypeptide chains, and differences in the terminations of the helicaldomain directly lead to distinct types of collagens. Its general groups include fibrillar collagens,FACIT (Fibril Associated Collagens with Interrupted Triple Helices), FACIT-like collagen, beadedfilament collagen, basement membrane collagen, short-chain collagen, transmembrane collagen, andunclassified collagen [29,33]. The length of the helix and portions of non-helical components aredifferent depending on the type of collagen.

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Numerous studies on collagen have revealed that collagen type I is the most ubiquitous form ofcollagen which belongs to the fibrillar group [29,30]. Fibril forming collagen or fibrillar collagen issynthesized in the form of soluble precursor molecules (procollagen) by the process of fibrillogenesis.Each polypeptide chain is involved in the synthesis process and consists of N- and C- propeptides ateach terminal position of the triple helix [22]. The fibrils produced have a visible banding, a direct resultof the aggregating pattern of collagen. The stability of the fibrillar collagen depends on non-reduciblecovalent cross-links in the triple helix [34].

The name FACIT collagen implies that the association of fibrils is interrupted by non-helicaldomains. They are linked with the surface of collagen fibrils and the collagenous structure is disturbedby non-helical domains. Wang et al. [22] further described that the C-NC domain in FACITs is shortcompared to fibril forming collagens. Collagen types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII belongto the FACIT group [32]. Wu, Woods, and Eyre [35] explained this scenario by depicting the structureof type IX collagen that lies anti-parallel to type II fibrils. Moreover, primary sequences of some FACITcollagens share similarities with fibrillar collagens [29].

The beaded filament collagen molecules assembled without undergoing the cleaving of terminalregions and the formation of the bead region in collagen filaments are facilitated by these uncleavedregions [29,32,35]. The most characteristic feature of this subgroup is having large N- and C-terminals.For example, type VI is having large N- and C-terminals even in their short triple- helical domains [29,30,36].Furthermore, only type VI collagen belongs to the subgroup of beaded filament collagen [29].

The basement membrane and associated collagen are categorized under non-fibrillar collagen.They can be found mostly in tissue boundaries, which facilitate molecular filtration by forminga connected network, especially in basement membranes [21,22]. Apart from tissue boundaries,non-fibrillar collagen can be found in cavities of the epithelial lining, endothelium in the interior bloodvessels, fat, muscle, and nerve cells. Based on the electron microscopic images, collagen IV belongsto the non-fibrillar collagen subgroup that appear as thin sheets and its molecules are relatively longcompared to the fibrillar collagen [30]. Anchoring fibrils collagen VII are considered as essential forfunctional integrity [22]. Short-chain collagens are described as mesh forming collagen and are locatedin underlying endothelial cells. Some of the short-chain collagens are also present in mineralizingcartilage [28]. The short-chain collagen possesses a shorter triple-helical region (half of the lengthof fibrillar collagen). Type VIII and X are categorized under the subgroup of short-chain collagen.Among them, type VIII collagen involves the proliferation of cells as a growth enhancer [29].

The transmembrane collagens function as cell surface receptors as well as matrix componentsinvolved in adhesion [29,30,37]. Moreover, they possess a relatively long but interrupted triple-helicaldomain with a short N terminal domain [38]. Type XIII, XVII, XXIII, XXV, and other collagen-likeproteins are categorized under transmembrane collagens [37,38].

2.2. Nomenclature, Types, and Classifications

After discovering type I, II, and III collagen, further research studies were evoked on theidentification of possible molecular types of collagen. However, expanded studies indicated thattype III collagen molecules also contained type I collagen and both types together could form mixedfibrils. This observation affected the terminology that existed then and became more complicatedafter the identification of type IV collagen [39]. Due to the variations of their histology, it was agreedto give a type number so, they are numbered with Roman numerals (I-XXIX) while polypeptidechains are named using α chains with Arabic numerals (α1, α2, α3, etc.). For instance, type I collagenwith identical α1(I) chains and one chain α2(I) and the nomenclature for type I collagen is [α1(I)]2

α2(I) [38,39]. Table 1 represents the some of the common types of collagen with their nomenclatureand distribution.

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Table 1. Common types of collagen.

Collagen Type Chains Sub Family Distribution

I α1(I)α2(I) Fibrillar collagen Skin, tendon, bone, dermis,

intestine, uterus

II α1(II) Fibrillar collagen Hyaline cartilage, vitreous,nucleus pulposus

III α1(III) Fibrillar collagen Dermis, intestine, large vessels,heart valve

IV

α1(IV)α2(IV)α3(IV)α4(IV)α5(IV)α6(IV)

Basement membrane andassociated collagen Basement membranes

Vα1(V)α2(V)α3(V)

Fibrillar collagen Cornea, placental membranes, bone,large vessels

VIα1(VI)α2(VI)α3(VI)

Beaded filament forming collagen Descement’s membrane, skin,heart muscles

VII α1(VII) Basement membrane andassociated collagen

Skin, placenta, lung,cartilage, cornea

VIII α1(VIII)α2(VIII) Short chain collagen Produced by endothelial cells,

descemet’s membrane

IXα1(IX)α2(IX)α3(IX)

Fibril associated andrelated collagen Cartilage

X α1(X) Short chain collagen Hypertrophic andmineralizing cartilage

XIα1(XI)α2(XI)α3(XI)

Fibrillar collagen Cartilage, intervertebral disc,vitreous humor

XII α1(XII) Fibril associated andrelated collagen

Chicken embryo tendon, bovineperiodontal ligament

XIII α1(XIII) Trans membrane collagens andcollagen like proteins Cetal skin, bone, intestinal mucosa

Source: Adapted from [40,41].

3. Sources of Collagen

As the major structural proteins are in the skin and bones of most animals, collagen accountsfor 30% of the total body protein [10]. The most common raw materials for collagen production areobtained from the slaughterhouse by-products, including hides, bones, tendons, and cartilages, orrecombinant collagen. At the industrial-scale production, animals such as bovine and pigs are used asprimary sources of collagen [9]. Figure 3 represents the most common sources of collagen. However,the outbreak of prion diseases such as bovine spongiform encephalopathy (BSE) resulted in somebarriers for using bovine collagen whereas swine flu has limited the use of porcine collagen [42].

In addition, due to various religious constraints, porcine or mammalian collagen for thedevelopment of kosher and halal products is limited [10,21,22]. Apart from the widely used species,several studies (Table 2) have extracted collagen from chicken [43], kangaroo tail [44], rat tail tendon [45],duck feet [46], equine tendon [33], alligators bone [47], birds’ feet [48–50], sheep tendon [51–55],and frog skin [56], while some studies have focused on using recombinant human collagen [20].

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The high pathological risk for transmitted diseases and complicated extraction process have limited theapplicability of using land animal collagen and created a growing concern towards finding alternativesources for collagen. The two primary sources of industrial collagen, including land animal by-productsand marine organisms, are described in the following subsections.

Table 2. Alternative land animal sources for bovine and porcine collagen.

Source Extraction Method Purpose of Extraction Reference

Chicken feet

Acid extraction Optimization of extraction condition [43]

Enzyme extraction Determination of pepsin digestion effect onthe properties of extracted collagen [48]

Acid extraction Preparation of edible films [57]

Enzyme extraction (usingpapain and pepsin)

Isolation and characterization of chickenfeet originated collagen [58]

Acid extraction Use of chicken feet for protein films [59]

Alkali, acid, and enzymeextraction

Identification of best method of collagenextraction method and characterization of

chicken feet collagen[50]

Enzyme extractionOptimization of extraction process andsynthesis of chicken feet collagen based

biopolymeric fibers[60]

Rat tail tendon Acid extraction Preparation of type I collagen for tissueengineering applications [45]

Alligator bone Acid and enzyme assistedextraction

Determination of biochemical properties ofalligator bone collagen [47]

Silky fowl feet Combination of acid andenzyme extraction

Identification of best combination for highquality collagen extraction method [49]

Ovine tendon Acid extractionDetermination of the biocompatibility ofovine tendon originated collagen with

human dermal fibroblast[51]

Acid extraction

Determination of the biocompatibility ofovine tendon originated collagen with

human dermal fibroblastImprove the mechanically strong ovine

tendon originated collagen for tissueengineering purposes

[52]

Acid extractionCharacterization and fabrication of thin

films from ovine tendon collagen for tissueengineering applications

[53]

Acid extractionInvestigation of attachment, proliferation,and morphological properties of human

dermal fibroblasts on ovine tendon collagen[54]

Duck feet

Acid extraction Investigation of physicochemical propertiesof collagen derived from duck feet [46]

Acid extractionDetermination of feasibility of using duck

feet collagen in improving physicochemicalproperties of surimi

[61]

Kangaroo tail Acid extraction Identification of alternative collagen sourcesfor pre-clinical models for cell biology [44]

Sheep bone Acid extraction Determination of effect of different collagenextraction protocols [55]

Equine tendon Acid extractionEvaluation of the effects of differentextraction methods on the collagen

structure of equine tendons[33]

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Figure 3. Popular sources of collagen.

In addition, due to various religious constraints, porcine or mammalian collagen for the development of kosher and halal products is limited [10,21,22]. Apart from the widely used species, several studies (Table 2) have extracted collagen from chicken [43], kangaroo tail [44], rat tail tendon [45], duck feet [46], equine tendon [33], alligators bone [47], birds’ feet [48–50], sheep tendon [51–55], and frog skin [56], while some studies have focused on using recombinant human collagen [20]. The high pathological risk for transmitted diseases and complicated extraction process have limited the applicability of using land animal collagen and created a growing concern towards finding alternative sources for collagen. The two primary sources of industrial collagen, including land animal by-products and marine organisms, are described in the following subsections.

Figure 3. Popular sources of collagen.

3.1. Land Animal By-Products

In recent decades, inedible animal by-products are utilized to produce fertilizers, minerals, fattyacids, vitamins, protein hydrolysates, and collagen [62]. Bovine collagen is the primary source for theindustrial collagen used in medicine, cosmetics, and other non-biomedical applications [63]. Sterilizedpurified collagen from cow skin is used as injectable bovine collagen [64]. Apart from BSE risk, around3% of the population is allergic to bovine collagen, which hinders its usage [20].

Skins and bones of pigs are used to extract porcine collagen [20]. Pig rind is famous for processingfood products such as sausage casings and edible films. Moreover, porcine collagen is used as a dermalsubstitute in the medical field as they are used widely as implants for reconstructive surgery [65].Pig hides are used to extract porcine type I collagen and share similar properties to human collagen,hence it has a wide range of application in both medical and food industries [65–67].

Collagen extraction from poultry by-products such as skin, bones, and cartilage from chickenhas also been reported. However, the usage was limited due to the occurrence of avian influenza [68].The mammalian collagen is preferred in the industrial level applications over avian collagen. The limitedapplications of avian collagen correlate with the expensive and complicated extraction process.

3.2. Marine Organisms

The marine-derived collagen is a promising alternative due to the occurrence of foot-and-mouthdisease (FMD), BSE, and avian influenza like diseases, as well as religious and social constraints [69,70].Several comprehensive reviews on marine-derived collagen and their application in various fieldshave appeared [17,20,71]. Recently, collagen from various marine organisms such as poriferans,

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coelenterates, annelids, mollusks, echinoderms, and crustaceans has been extensively investigated(Figure 4).

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3.1. Land Animal By-Products

In recent decades, inedible animal by-products are utilized to produce fertilizers, minerals, fatty acids, vitamins, protein hydrolysates, and collagen [62]. Bovine collagen is the primary source for the industrial collagen used in medicine, cosmetics, and other non-biomedical applications [63]. Sterilized purified collagen from cow skin is used as injectable bovine collagen [64]. Apart from BSE risk, around 3% of the population is allergic to bovine collagen, which hinders its usage [20].

Skins and bones of pigs are used to extract porcine collagen [20]. Pig rind is famous for processing food products such as sausage casings and edible films. Moreover, porcine collagen is used as a dermal substitute in the medical field as they are used widely as implants for reconstructive surgery [65]. Pig hides are used to extract porcine type I collagen and share similar properties to human collagen, hence it has a wide range of application in both medical and food industries [65–67].

Collagen extraction from poultry by-products such as skin, bones, and cartilage from chicken has also been reported. However, the usage was limited due to the occurrence of avian influenza [68]. The mammalian collagen is preferred in the industrial level applications over avian collagen. The limited applications of avian collagen correlate with the expensive and complicated extraction process.

3.2. Marine Organisms

The marine-derived collagen is a promising alternative due to the occurrence of foot-and-mouth disease (FMD), BSE, and avian influenza like diseases, as well as religious and social constraints [69,70]. Several comprehensive reviews on marine-derived collagen and their application in various fields have appeared [17,20,71]. Recently, collagen from various marine organisms such as poriferans, coelenterates, annelids, mollusks, echinoderms, and crustaceans has been extensively investigated (Figure 4).

Figure 4. Marine sources of collagen.

The unique characteristics of marine collagen as a biomaterial with significant biocompatibility and biodegradability has been favored in many industrial applications over other alternate sources [69,72]. Mainly, marine by-products have been exploited to recover collagen and other collagen-derived biomaterials through a combination of different bioprocessing methods [73]. These include Japanese sea bass skin [74], clown feather back skin [75], bladder of yellow fin tuna [76], fin, scales,

Figure 4. Marine sources of collagen.

The unique characteristics of marine collagen as a biomaterial with significant biocompatibility andbiodegradability has been favored in many industrial applications over other alternate sources [69,72].Mainly, marine by-products have been exploited to recover collagen and other collagen-derivedbiomaterials through a combination of different bioprocessing methods [73]. These include Japanesesea bass skin [74], clown feather back skin [75], bladder of yellow fin tuna [76], fin, scales, skins,bones, and swim bladders of big head carp [77], skin and bone from Japanese seerfish, cartilage fromsturgeon and sponges, sea urchin [78], octopus [79] squid [80], cuttlefish [81], sea anemone [82], andsea cucumbers for extraction of marine collagen [83]. Particularly, collagen type I was extracted fromthe skin of silver carp [84], Japanese sea-bass [74], mackerel [85], bullhead shark [86], and sole fish [87]as well as from the bones of skipjack tuna [88], and scales of Nile tilapia [89].

Significant differences in the amino acid composition of collagen from various fish species areresponsible for their unique characteristics [69]. Most of the fish collagen contains a lower proportion ofhydroxyproline compared to mammalian and avian collagen. Consequently, their lower compatibilityto crosslinking and stability compared to other types of collagen has been reported [14,69,90]. However,the content of hydroxyproline also depends on the habitat of fish species [91]. Moreover, the thermalstability of the collagen extracted from warm water species is found to be higher than cold waterspecies [91].

The marine sources of collagen have received increasing attention due to their availability, easyprocessing techniques, safety (free of zoonosis), environmentally friendly extraction procedures, lowmolecular weight, less religious and ethical barriers, minor regulatory and quality control problems, anegligible amount of biological contaminants and toxins, low inflammatory response and excellentmetabolic compatibility [20]. However, most studies have been conducted to identify the potentialuses of collagen derived from marine vertebrates but reports on marine invertebrates are scarce [4,17].Thus, current research interest is directed towards the use of marine invertebrates as potential sourcesof collagen, particularly for biomedical applications. Recent investigations have been concentrated onjellyfish [92] sponges [93], mussels [4,94], and sea cucumber [14,16,83,95–105] as potential candidatesfor producing marine-derived collagen.

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3.3. Sea Cucumber as A Source of Collagen

Among the various bioactive compounds derived from sea cucumber, collagen plays a vital role.Primary intensive research on sea cucumber collagen has been initiated in the early 1970s by Eyre andGlimcher [106] and Matsumura, Shinmei, and Nagai [107]. Eyre et al. [108] studied the comparativebiochemistry of the collagen crosslinks using sea cucumber Thyone briarius, a sponge Haliclona oculata,and sea urchin Strongylocentrotus droebachensis and reported the evidence for glycosylated crosslinksin collagen derived from the body wall of sea cucumber Thyone briarius. Matsumura et al. [107]then focused on the purification of collagen from sea cucumber Stichopus japonicus by disaggregatingthe connective tissue of body wall followed by the morphological study of the isolated collagenfibrils. Furthermore, the most extensive research studies were focused on the molecular structureand functional morphology of Cucumaria frondosa which led to a series of discoveries on the covalentcomposition and growth of collagen fibrils in the same species [109–111]. The dermal glycoproteinstiparin was identified as the main factor responsible for the aggregation of collagen fibrils from thedermis of sea cucumber Cucumaria frondosa [112] and Trotter et al. [113] characterized a sulphatedglycoprotein, which inhibited fibril-aggregating activity.

Thurmond and Trotter [114] further investigated the morphology and biomechanics of themicrofibrillar network of collagen derived from sea cucumber Cucumaria frondosa dermis and reportedsimilar morphological characteristics with fibrillin microfibrils of vertebrates. Most of the earlyinvestigations of the sea cucumber collagen fibrils contributed to recent developments of the researchrelated to collagen and other bioactivities from sea cucumber. Table 3 provides a cursory account ofrecent studies related to sea cucumber collagen.

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Table 3. Recent studies on sea cucumber collagen.

Sea Cucumber Species Focus of Study Major Findings Reference

Stichopus japonicus Chemical composition and subunit structure of collagen Collagen was comprised of 2 distinct subunits (α1 and α2 and rich inglutamic acid compared to other fibrillar collagen [14]

Characterization and subunit composition of collagen Pepsin solubilized collagen resembled type I collagen and its amino acidcomposition was different from vertebrate collagen. [16]

Changes of collagen during cooking Crude collagen fibers were more susceptible to heat treatment compared topepsin-solubilized collagen [115]

Identification of physicochemical properties and radicalscavenging capacities of pepsin-solubilized collagen

Extracted collagen maintained intact triple-stranded helices and highmoisture retention and absorption capacities as well as exhibiting better

radical scavenging ability compared to vitamins C and E.[95]

Wound-healing effects on human keratinocyte (HaCaT)cell line of pepsin-solubilized collagen

Pepsin-solubilized collagen has the potential to use as an alternativemammalian collagen in the nutraceutical and pharmaceutical industries [96]

Effect of autolysis of intact collagen fibers related to thedistributions of cathepsin L

Lysosomal cathepsin L degrades the collagen fibers and speed and degree ofautolysis is negatively correlated with the density of collagen. [116]

Structural characteristics of sea cucumber collagen fibersin the presence of endogenous cysteine proteinases

Collagen fibrils disaggregated into collagen fibrils by cysteine proteinasesand the structural disorder of the native collagen fibers increased due to

cysteine protease.[102]

Structural and biochemical changes ofcollagen related to autolysis

Collagen fibers and microfibrils gradually degraded with the autolysis andstructural damage was less in monomeric collagen compared to other

structural elements[103]

Structural and thermal properties ofsea cucumber collagen

Distance between adjacent molecular chains in collagen molecules wasdecreased and CO2, NH3, H2O, CH4, NO2 and HCN gases released during

the heat treatment[117]

Enzymatic hydrolysis of collagen to determine thestructural changes of collagen fibrils

Collagen fibers were partially degraded into collagen fibrils by enzymatic(trypsin) treatments [118]

Investigate the effect of collagenase type I on thestructural features of collagen fibers

Collagenase was responsible for partial depolymerization of collagen fibersinto fibrils, uncoiled the fibrils, degrade monomeric collagen [119]

Parastichopus californicus Purification and characterization of pepsin-solubilizedcollagen from skin and connective tissue

Collagen extracted from skin and connective tissue contains type I collagenwith three α1 chain. Amino acid composition is different from the

mammalian type I collagen[120]

Bohadschia spp. Analysis of isolated pepsin-solubilized collagen Type I collagen was identified with three α1 chain [121]

Stichopus vastus Isolation and characterization ofpepsin-solubilized collagen

Purified collagen belongs to type I collagen contains three α1 chain withtriple helical structure [99]

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Table 3. Cont.

Sea Cucumber Species Focus of Study Major Findings Reference

Molecular mass distribution, amino acid composition andradical-scavenging activity of collagen hydrolysates

prepared from isolated collagen

β and α1 chains of the collagen were hydrolyzed by trypsin and molecularmass distribution ranged from 5 to 25 kDa. Hydrolysates contains highglycine, alanine, glutamate, proline and hydroxyproline residues and

showed significant radical scavenging activity

[122]

Physicochemical and biochemical properties of pepsinsolubilized collagen

Glycine was the predominant amino acid present in purified collagen thatpossessed high moisture absorption and retention capacity [122]

Identification of Angiotensin I converting enzyme (ACE)inhibitory and radical scavenging activities from

collagen hydrolysates

Novel bioactive peptides generated by optimized trypsin hydrolysis havethe potential to use as ACE inhibitors and radical scavenging agents. [100]

Holothuria parva Purification and characterization ofpepsin-solubilized collagen

Isolated collagen constituted three α1 chain and was rich in glycine, proline,alanine and hydroxyproline [98]

Stichopus monotuberculatus Isolation and characterization ofpepsin-solubilized collagen Isolated collagen was classified as type I collagen consisted of three α1 chain [101]

Holothuria scabra Determination of nano-collagen quality and extraction ofacid solubilized collagen

Extracted acid solubilized collagen had significant effect on physicochemicalcharacteristics of nano-collagen particles [123]

Australostichopus mollis Biochemical composition of isolated collagenType I collagen was present with α1 and α2 chains, α chain dimers, β chains,

and γ components. Most abundant amino acids were glycine, alanine,threonine, serine, and proline.

[124]

Holothuria leucospilota In vitro activity of anti-tyrosinase and anti-elastaseactivity of isolated collagen

Isolated collagen exhibited weak anti-tyrosine activity and moderateanti-elastase activity [125]

Acaudina leucoprocta Extraction methods to remove heavy metals from theisolated collagen

Pepsi- solubilized collagen showed two isoforms and amount of heavymetals present in the collagen were below the contaminant limit [126]

Acaudina molpadioides Preparation and characterization of antioxidativepeptides from collagen hydrolysates

Collagen peptides which showed highest antioxidant activity were rich inhydrophobic acid residues. [127]

Stichopus vastus andHolothuria atra

Comparison of partial characteristics of two differentsea cucumbers No significant difference in amino acid composition, yield, or whiteness [128]

Apostichopus japonicus Type of constituent collagen using proteomics andbioinformatic strategies

Heterogenicity of the sea cucumber collagen fibrils was revealed for the firsttime that provides novel insight into the composition of sea

cucumber collagen[104]

Analysis of the effect of epigallocatechin gallate (EGCG)on preserving molecular structure of collagen fibers

during heating

EGCG protects the structure of crude collagen fibers in a dosage dependentmanner and effects hydrogen bonds on the collagen which promotes

protein aggregation[105]

Holothuria cinerascens Potential application of collagen inmoisturizing cosmetics

Collagen showed better moisture retention and moisture absorptioncapacity. Abundant hydrophilic groups in collagen increases their ability for

cosmetic formulations[83]

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Sea cucumber research interests have been mainly focused on cultivation and bioactive molecules.Most of the research conducted on bioactive ingredients from sea cucumber has centered aroundproteoglycan and collagen [83]. The main edible portion of sea cucumber is the body wall composed ofmutable connective tissue (MCT) with scattered cells [114]. The structural components of MCT consistof collagen, proteoglycan, and glycoprotein [118]. These assembled components form collagen fibrils,collagen fibers, and microfibrils. Among them, the majority of total body wall protein are comprisedof insoluble collagen fibrils. Collagen fibers are surrounded and separated from the microfibrillarnetwork in MCT and this network maintains the organization while providing a long-range restoringforce [120].

The most abundant type of collagen found in sea cucumber is type I collagen and collagen fibrils ofechinoderms are symmetrically spindle-shaped and short in length [113,120]. Moreover, at the molecularlevel, they are considered as bipolar collagen fibrils with surface associated proteoglycans [113].Covalent crosslinks providing stabilization to collagen are internally present and similar to themammalian collagen. Besides, absence of permanent crosslinks in the structure improves the isolationof collagen fibrils in their intact form [98,113]. It also helps to slide pass one another during lengtheningand shortening of the tissue [114]. The solubilized collagen from the body wall of sea cucumber(Stichopus japonicus) has distinct subunit structure of (α1)2 α2 and are rich in glutamic acid. Thermaldenaturation of this type of collagen may impart unique textural properties [14]. A recent study on themolecular composition of collagen fibrils isolated from sea cucumber Aposticopus japonicus revealedthat collagen fibrils are heterotypic containing two clade A, one clade B fibrillar collagens, and twoFACIT collagens [104]. Fibrillar collagen α chains may be classified in to three clades according to theirevolutionary steps. Clade A consists of α1(I), α2(I), α1(II), α1(III), and α2(V) chains; clade B containsα1(V), α3(V), α1(XI), and α2(XI) chains while clade C includes α1(XXIV) and α1(XXVII) chains [129].

Tian et al. [104] also reported the heterogenicity exhibited in the pepsin-solubilized collagenisolated from Aposticopus japonicus for the first time. Their novel findings on subunit compositionsand constituents of sea cucumber collagen were, however, contradictory to the previousstudies [14,16,98,99,101,102,109,115,120]. Most of the previous studies focused on pepsin-solubilizedcollagen (PSC), and structure analysis was conducted using sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE). However, Tian et al. [104] used proteomic techniques and bioinformaticmethods to analyze the constituents present in sea cucumber collagen. According to the phylogeneticanalysis of identified collagen sequences revealed that reported sea cucumber collagen sequencesdid not belong to the branches of typical collagens. The authors concluded that the heterogenicand complex nature of the sea cucumber collagen is complicated and needs extensive investigations.Thus, previously reported studies on SDS-PAGE analysis are not considered adequate to conclude thefundamental molecular structure of collagen [104].

4. Characteristics and Properties of Collagen Type I

Hierarchical structures configured from the fibrillar collagen include collagen α chains,tropocollagen, collagen fibril and collagen fibers [104,130]. The most abundant structural collagen inmost tissues is the fibrillar type I collagen [131]. Primarily, type I collagen (Figure 5) is present in fibrilsurface as well as notably in connective tissues of the skin and bone and has distinct structural featuresincluding wide distribution of fibril diameters and high internal crystallinity [64]. Type I collagen fibrilis formed by two equivalent α1 and one α2 polypeptide chains and composed of 1.1 × 300 nm sizecollagen molecules [130,132]. The two α chains form peptide chain dimer referred to as β-peptidechain while three α chains form γ-peptide chain. Each polypeptide chain weighs around 100 kDa andis comprised of 1052 amino acid residues.

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Figure 5. Chemical structure of collagen type I-Primary amino acid sequence.

The γ-peptide chain is referred to as the tropocollagen molecule. Terminal extensions of type I tropocollagen included 139 amino acids and COOH which weigh 20,000 and 35,000 Da, respectively [41,64,132]. Type I collagen contains high amounts of proline and hydroxyproline compared to other types of collagen [41,132]. Moreover, collagen type I is considered as a glycoprotein and composed of less than 1% carbohydrate, including either single galactose unit or disaccharide of galactose and O-glycosidically attached via hydroxylysine residues as sugar components [41]. Table 4 summarizes some of the distinct characteristics of sea cucumber type I collagen reported in literature compared to the mammalian collagen.

Table 4. Distinct characteristics of sea cucumber collagen compared to mammalian collagen.

Characteristics Sea Cucumber Derived Collagen Mammalian Collagen Reference

Abundant type Type I collagen Type I collagen [14,18,113]

Differences in amino acid

composition

Low hydroxyproline content, high glutamic and

aspartic acid residues

High hydroxyproline content, low glutamic acid and aspartic acid

residues

[14,16,99,101,120]

Covalent cross links

Internally present and provide stabilization to the

molecule

Internally present and provide stabilization to

the molecule [109–111]

Thermal stability

Low thermal stability with low denaturation

temperature compared to mammalian collagen

High thermal stability compared with high

denaturation temperatures

[98,101,120,122]

Resistance to protease digestion

Relatively low Relatively high [99]

Gel forming ability Comparatively low Comparatively high [99]

Moisture absorption ability Relatively high Relatively high [8,95]

Figure 5. Chemical structure of collagen type I-Primary amino acid sequence.

The γ-peptide chain is referred to as the tropocollagen molecule. Terminal extensions oftype I tropocollagen included 139 amino acids and COOH which weigh 20,000 and 35,000 Da,respectively [41,64,132]. Type I collagen contains high amounts of proline and hydroxyprolinecompared to other types of collagen [41,132]. Moreover, collagen type I is considered as a glycoproteinand composed of less than 1% carbohydrate, including either single galactose unit or disaccharide ofgalactose and O-glycosidically attached via hydroxylysine residues as sugar components [41]. Table 4summarizes some of the distinct characteristics of sea cucumber type I collagen reported in literaturecompared to the mammalian collagen.

Table 4. Distinct characteristics of sea cucumber collagen compared to mammalian collagen.

Characteristics Sea Cucumber DerivedCollagen Mammalian Collagen Reference

Abundant type Type I collagen Type I collagen [14,18,113]

Differences in amino acidcomposition

Low hydroxyproline content,high glutamic and aspartic

acid residues

High hydroxyprolinecontent, low glutamicacid and aspartic acid

residues

[14,16,99,101,120]

Covalent cross linksInternally present and

provide stabilization to themolecule

Internally present andprovide stabilization to

the molecule[109–111]

Thermal stability

Low thermal stability withlow denaturation

temperature compared tomammalian collagen

High thermal stabilitycompared with high

denaturationtemperatures

[98,101,120,122]

Resistance to proteasedigestion Relatively low Relatively high [99]

Gel forming ability Comparatively low Comparatively high [99]Moisture absorption ability Relatively high Relatively high [8,95]

4.1. Thermal Stability

The term ‘stability’ in protein ultimately refers to sustaining the significant structure of proteinunder extreme conditions [133]. The type I collagen is thermodynamically stable compared to kineticstability [134]. However, the triple helical structure is susceptible to heat and particularly type I collagenhas low thermal stability [135]. Moreover, thermal stability is considered as one of the significantfactors for determining the potential application of collagen [136]. The amino acid composition directlyinfluences the physical and chemical properties of collagen, including thermal stability. The stabilityof the triple helix structure is improved by the hydrogen bonds formed by the hydroxyl groups ofhydroxyproline [101]. The amino acid content influences the thermal stability of type I collagen.The molecular structure of collagen mainly depends on the secondary structure of the polypeptidechain [137]. Pyrrolidine rings of proline and hydroxyproline are mainly responsible for the uniquestructure of collagen and the helical stability is directly proportional to the amino acid content [101].

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The thermal stability of collagen, as any other protein, is often described in accordance with itsdenaturation temperature (Td) and the maximum transition temperature (Tm) [101,133]. Td denotesthe temperature at which the triple-helix structure of collagen disintegrates into random coils, andwhen it reaches Tm, half of its triple helix is degraded to obtain the maximum transition temperatureof the collagen [101,138]. The transition temperature correlates with collagen stability and durabilityof collagen-based biomaterials [48]. Determination of the thermal stability using differential scanningcalorimetry (DSC) thermogram will be discussed in later sections of this review.

Numerous studies have been conducted to elucidate the thermal stability of isolated collagenfrom sea cucumber. Thermal behavior of the sea cucumber derived collagen was mostly comparable totype I bovine collagen. Isolated collagen from sea cucumber (Stichopus monotuberculatus) exhibitedlower (30.2 ◦C) Tm than calfskin collagen (35 ◦C) and positive relationship existed among Tmvalue and imino acid content [101]. Adibzadeh et al. [98] reported a similar trend for the pepsinsolubilized collagen from sea cucumber Holothuria parv and showed lower thermal stability comparedto type I bovine collagen and porcine skin collagen. Thermal behavior of the collagen isolated fromStichopus japonicus [16], Stichopus vastus [122], and Parastichopus californicus [120] further explainsthe lower transition temperatures irrespective of the species, and shows that sea cucumber derivedcollagen possesses weak thermal stability compared to mammalian collagen. This may be due to thefactors influencing the thermal stability of collagen originating from vertebrates and invertebrates [48].Besides the amino acid composition (especially amount of amino acid residues), the environment andbody temperature of the animal is a determinant factor for thermal sensitivity of collagen fibrils [101].Lin and Liu [48] revealed that marine collagen has a lower denaturation temperature in contrast withcollagen derived from land animals.

It is noteworthy that thermal stability has a direct relation with the amino acid composition asmost of the isolated sea cucumber collagen is rich in hydroxyproline and proline [16,101,120]. However,most studies on sea cucumber collagen have been focused on pepsin solubilized collagen (PSC), andthese PSCs do not represent the native structure of the dermic collagen [138]. Qin et al. [138] studiedthe thermal behavior of insoluble collagen fibrils and PSC from sea cucumber (Stichopus japonicus).According to their findings, helical structures of insoluble collagen fibrils are more stable than those ofpepsin soluble collagen. The difference is mainly due to the removal of cross-linkages in the telopeptideregion of native collagen fibrils. Therefore, insoluble collagen fibrils show higher thermostabilitycompared to PSC [138].

Furthermore, thermal stability plays a significant role in the sea cucumber processing industry [117].Few studies have examined the thermal behavior of collagen during processing. One study investigatedthe thermal denaturation of crude collagen fibers (CCF) and PSC during cooking [115]. CCF wasmore thermostable than the PSC at different tested cooking temperatures of 40–100 ◦C. Besides theDSC method, a Fourier transform infrared (FTIR) method was also employed to analyze the thermalproperties of collagen. Si et al. [117] combined the FTIR and thermogravimetric analysis (TGA) todetermine the thermal degradation mechanism of sea cucumber (Stichopus japonicus). The thermaldegradation activation energy of sea cucumber collagen revealed that higher treatment temperatureswere not applicable for cooking or processing of sea cucumbers. In addition, it may have adverseeffects on the nutritional value of sea cucumber due to the denaturation of proteins [117].

Furthermore, previous studies have reported the effect of thermal treatment on collagen fibrils inrelation to oxidation [139]. A recent study investigated the molecular structure of collagen isolatedfrom Apostichopus japonicus during thermal treatment in the presence of (-)-epigallocatechin gallate(EGCG) [105] and demonstrated that EGCG has the potential to enhance the thermal stability of crudecollagen fibrils by neutralizing the effect of heat-induced radicals (hydroxyl radical) and protect themacromolecular structure of crude collagen in a dose-dependent manner [105].

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4.2. Enzymatic Resistance and Digestion

The biomaterial market prefers a higher enzymatic resistance collagen due to its higherdurability [48]. Enzymes that could break the triple helix of collagen are known as collagenolyticenzymes [140]. As collagen plays a role as one of the primary structural body proteins, it has peculiarresistance for neutral proteases [41]. Degradation of collagen molecules starts from the exteriorby binding of collagenase to the triple helix near the surface and proceeds with the progression ofdegradation in the interior of molecules when exposed to the collagenase enzymatic action [41]. Severalstudies have indicated that collagenase is capable of cleaving all three α- chains of type I collagen ata single site and results in the formation of fragments about three quarters and one-quarter of theoriginal size of the molecules [140].

Liu et al. [119] investigated the role of collagenase type I on the structural features of collagenfibers from sea cucumber (Stichopus japonicus). Collagenase partially depolymerized collagen fibers intofibrils of Stichopus japonicus by influencing proteoglycan interfibrillar bridges. Furthermore, collagenasehas the potential of degrading the monomeric collagen [119]. These findings provide evidence aboutthe role of collagenase in the autolysis of sea cucumber. The autolysis of sea cucumber is due to anactivation process of endogenous proteinases such as cysteine proteinase, serine proteinase, and matrixmetalloproteinases. Proteases responsible for autolysis are involved in the depolymerization andunfolding of collagen fibrils [141,142]. Collagenase enzyme represents the matrix metalloproteinasesgroup, and collagenase from the dermis of Stichopus monotuberculatus was reported to hydrolyzethe triple-helix of collagen [142]. These observations are in accordance with the conclusions ofLiu et al. [119], implying that endogenous matrix metalloproteases have the ability of digest themacromolecular and monomeric collagens from sea cucumber.

Moreover, serine collagenases are considered as specific collagenase enzymes that can break downthe substrate under any conditions. After the initial cleavage of collagen, the polypeptide chains arefurther degraded by other protease enzymes such as gelatinases and non-specific proteinases [41].Trypsin belongs to the group of a serine protease and used as a hydrolyzing agent to determine the roleof serine proteases in the autolysis process of sea cucumber collagen [118]. The results showed thattrypsin has the potential to partially disintegrate the collagen fibrils as well as cleave the interfibrillarproteoglycan bridges with a lower effect on monomeric collagen. Liu et al. [102] studied the effect ofendogenous cysteine proteinases on collagen fibers from Stichopus japonicus and reported changes inthe microstructure of collagen fibrils. Endogenous cysteine proteinases degraded the interfibrillarproteoglycan bridges and increased the structural disorder of fibrillar collagen. Investigations ofcysteine proteases, including cathepsins K and L, demonstrated the disintegration of the collagenfibrils caused by the activity of cathepsin L-proteinase on proteoglycan networks [141].

In contrast, several studies have found that some collagens extracted from different species can havehigher stability even in the collagenase solution. Lin and Liu [48] reported that the porcine skin type Icollagen was more stable compared to the other collagen species, while Angele et al. [143] indicatedthe higher stability of equine collagen compared to bovine collagen-based matrix. They concluded thatthe presence of a higher content of glycosaminoglycan was responsible for the stability of collagenas they blocked the cleavage sites of collagenase. Furthermore, Li and Liu [48] stated that marineanimal collagens were efficiently degraded by proteolytic enzymes and more sensitive to non-specificenzymatic hydrolysis compared to land animal collagens.

4.3. Isoelectric Point of Collagen

Chemical environment is crucial for the formation of collagen type I fibrils and pH plays a vital rolewhen determining the chemical properties. Proteins have zero electrostatic charges at their isoelectricpoint (pI) which represents the minimum solubility and maximum precipitation. Thus, pI of thecollagen is the primary determinant factor for its solubility. Moreover, pI indicates the pH value whichhas higher hydrophobic-hydrophobic interaction that leads to precipitation and aggregation of theprotein molecules [136].

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The pI of the collagen derived from sea cucumbers belongs to the acidic region. Zhu et al. [95]observed the isoelectric point of 4.14 for PSC from sea cucumber Stichopus japonicus. Similar findingswere reported for PSC from different sea cucumber species including Stichopus vastus, pI of 4.67 [122]and Stichopus monotuberculatus pI of 4.0 [101]. Furthermore, lower pI values correlated with the type ofamino acid residues present in the sample [101]. Most of the reported sea cucumber-derived collagenis abundant in glutamic and aspartic acids [14]. Friess [41] reported maximum collagen degradation atpH 4.4. Furthermore, neutral pH is important for collagenases enzyme to react with the triple helixstructure, specifically to cleave the band, which is three quarters away from the N- terminus of thenative helix. As the most significant parameter of the protein, the pI is related to the proportion ofacidic and basic amino acid residues present in protein [144].

4.4. Bioactive Properties of Sea Cucumber Collagen

Marine derived collagen is highly regarded as a valuable source with significant bioactiveproperties [12,15,100,125]. In terms of sea cucumber collagen, several studies have elucidated itsantioxidant potential associated with the radical scavenging capacities. Zhu et al. [95] investigated thepepsin soluble collagen from the body wall of sea cucumber Stichopus japonicus. It was demonstrated thathydroxyl radical scavenging ability and DPPH (2,2-diphenyl-1,1-picrylhydrazyl) radical scavengingactivity were significantly higher than those of vitamins C and E. The authors concluded that antioxidantactivities exerted by sea cucumber body wall was mainly attributed to collagens. Similar findingswere reported by Abedin et al. [100] on collagen hydrolysates prepared from sea cucumber Stichopusvastus using 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) activity assay. The authorsalso observed Angiotensin I converting enzyme (ACE) inhibitory potential of produced collagenhydrolysates [100]. Abdillah et al. [125] investigated the anti-tyrosine and anti-elastase activitiesof collagen extracted from body wall of sea cucumber Holothuria lecospilota and evaluated itspharmaceutical capacities. The study revealed the efficacy of sea cucumber collagen hydrolysatesrelated to their antiwrinkle capabilities. However, most of the pharmaceutical applications of marinederived collagen has been extensively studied for its potential as a biomaterial. Section 5.1 provides adetailed discussion of biomedical applications of sea cucumber collagen.

5. Industrial Applications

Collagen may be used in a wide range of applications in various fields due to its diversifiednature. The global demand for collagen has increased during the past few decades, with the boominginterest for using it as a biomaterial over other natural polymers and their synthetic analogs. Distinctphysicochemical properties of collagen expand its application in various fields, including biomedical,pharmaceutical, cosmetic and food industries (Figure 6). Marine collagen, as a promising alternativefor commonly used mammalian-derived collagen, has gained growing attention of both scientific andindustrial communities. However, reports on the industrial application of sea cucumber collagen arescarce compared to the mammalian collagen. Hence, the following sections include a general overviewof potential applications of type I collagen in biomedical and non-biomedical fields.

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Figure 6. Commercial products developed including sea cucumber collagen as a main ingredient. (Image courtesy: google image; manufactures’ websites).

5.1. Biomedical Applications

Collagen is considered as a successful biomaterial in medical applications, mainly due to its characteristics such as biodegradability and weak antigenicity. Its tensile and fibrous structure provides strength and elasticity to the skin in addition to strengthening blood vessels and tissue development [129]. In addition to these properties, the progress of use of collagen as a biomaterial is associated with numerous benefits such as high availability and efficient purification, biocompatibility and bioabsorbability, non-toxicity, synergism with bioactive compounds, compatibility with synthetic polymers, durability and persistence, ability for interaction with cell-matrix and platelets and most importantly fibril reformation [40,86]. Notably, the interest in collagen as a biomaterial depends upon its source and diverse morphologies [28].

Moreover, collagen has the ability of producing sheets, tubes, powders, fleeces, injectable solutions, and dispersions, which expand its usage in the medical sphere. These applications of collagen are tested in drug delivery systems in ophthalmology, wound and burn dressing, tumor treatment, and tissue engineering [41]. PSC from sea cucumber (Stichopus japonicus) has been investigated for its ability in wound-healing [96] and has shown increased cell migration and proliferation as well as wound-healing effects in human keratinocyte cell lines compared to conventional collagen [96]. These findings demonstrate the potential of sea cucumber collagen for use as an alternative collagen source in biomedical applications.

The collagen as a biomaterial can be used under different fields of applications such as tissue engineering, bone substitutes, eye implants, drug delivery matrix, gene delivery matrix, protein delivery matrix, and as a useful biomaterial which forms organoids or neo-organs in gene therapy [145]. Furthermore, the use of collagen in cosmetic surgeries is one of its significant applications due to commercial influence in the industrial sphere related to biocompatibility and safety [86]. Besides, collagen is often used as a hemostatic agent, and surgical suture ascribed to its shorter period of healing time over other traditional methods [41,86,145]. Furthermore, the use of marine collagen is becoming popular in the field of tissue engineering [146]. Carvalho et al. [147] studied the marine-derived type I collagen with receptors at the cell surface and its potential of involving cell adhesion, differentiation and growth and developed novel biomaterials using combination of other biopolymers with collagen. A recent study on Jellyfish collagen as biomaterial also demonstrated its

Figure 6. Commercial products developed including sea cucumber collagen as a main ingredient.(Image courtesy: google image; manufactures’ websites).

5.1. Biomedical Applications

Collagen is considered as a successful biomaterial in medical applications, mainly due to itscharacteristics such as biodegradability and weak antigenicity. Its tensile and fibrous structureprovides strength and elasticity to the skin in addition to strengthening blood vessels and tissuedevelopment [129]. In addition to these properties, the progress of use of collagen as a biomaterial isassociated with numerous benefits such as high availability and efficient purification, biocompatibilityand bioabsorbability, non-toxicity, synergism with bioactive compounds, compatibility with syntheticpolymers, durability and persistence, ability for interaction with cell-matrix and platelets and mostimportantly fibril reformation [40,86]. Notably, the interest in collagen as a biomaterial depends uponits source and diverse morphologies [28].

Moreover, collagen has the ability of producing sheets, tubes, powders, fleeces, injectable solutions,and dispersions, which expand its usage in the medical sphere. These applications of collagen aretested in drug delivery systems in ophthalmology, wound and burn dressing, tumor treatment,and tissue engineering [41]. PSC from sea cucumber (Stichopus japonicus) has been investigated forits ability in wound-healing [96] and has shown increased cell migration and proliferation as wellas wound-healing effects in human keratinocyte cell lines compared to conventional collagen [96].These findings demonstrate the potential of sea cucumber collagen for use as an alternative collagensource in biomedical applications.

The collagen as a biomaterial can be used under different fields of applications such as tissueengineering, bone substitutes, eye implants, drug delivery matrix, gene delivery matrix, proteindelivery matrix, and as a useful biomaterial which forms organoids or neo-organs in gene therapy [145].Furthermore, the use of collagen in cosmetic surgeries is one of its significant applications due tocommercial influence in the industrial sphere related to biocompatibility and safety [86]. Besides,collagen is often used as a hemostatic agent, and surgical suture ascribed to its shorter period of healingtime over other traditional methods [41,86,145]. Furthermore, the use of marine collagen is becomingpopular in the field of tissue engineering [146]. Carvalho et al. [147] studied the marine-derived type Icollagen with receptors at the cell surface and its potential of involving cell adhesion, differentiationand growth and developed novel biomaterials using combination of other biopolymers with collagen.A recent study on Jellyfish collagen as biomaterial also demonstrated its potential as a possible

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alternative to type I collagen source in fibrillar or nonfibrillar form for tissue engineering studies andindustrial use [148].

Echinoderm originated mutable collagenous tissues have the potential to develop the collagenbarrier-membranes for tissue regeneration applications [146]. The research on different echinodermmodels including sea cucumber (Holothuria tubulosa), sea urchin (Paracentrotus lividus), and starfish(Echinaster sepositus) substrates were used to determine their compatibility to exploit as collagen barriermembranes for guided tissue regeneration (GTR) process and demonstrated similar cell morphologyof all tested materials to commercially used bovine collagen substrate and echinoderm collagenoustissues [146]. Another specific advantage of echinoderm derived collagen is the tendency to maintainits original structure even after the extraction process [149]. The scaffolds, central fabrication to tissueengineering technology made of soluble jellyfish or squid collagen, have shown lower immunogenicityand higher cell viability compared to other biomaterials like bovine collagen [149,150]. Furthermore,collagen and its hydrolysates are used as a supplement for bone integrity, brittle nails treatment, andosteoarthritis pain [147–150].

In addition to the advantages linked to sea cucumber derived collagen, most Asians are stillconsidering sea cucumber as a traditional medicine for treating asthma, hypertension, rheumatism,and anemia [151]. Hence, application of collagen in the biomedical sector has expanded to the field ofpharmaceutical industries as well as in tissue engineering as injectable matrices, scaffolds for bonereconstruction, vascular, and cardiac reconstruction [19,20].

However, limitations of using marine collagen as biomaterial are inevitable. The diversities ofcross-link density, fiber size, and trace impurities are factors that hinder the use of isolated collagen.In addition, variability in enzymatic degradation rate and nature of hydrophilicity, production yieldover mammalian collagen and high cost associated with the preparation of type I collagen are consideredas some major drawbacks for the use of marine collagen [10,14,15,19,20]. Specifically, further in vitroand in vivo studies are necessary to extensively investigate the biocompatibility and immunogenicityof marine-derived collagen, including those from sea cucumbers for human clinical applications [146].

5.2. Non-Biomedical Applications

The industrial use of collagen in classical food, photographic, cosmetic and many other applications(involvement of leather production, producing gelatin-like hydrolysates) are mainly based on itsunique functional and technological properties [9]. The use of collagen as a source of glue has an8000 year history in protection of embroidered fabrics and tools and 4000 years as an adhesive used byEgyptians [8]

More recently, collagen is used widely in new food product development as a clinically provenhealthy nutritious food supplement. Collagen supplements are considered as an anti-aging agentwhich are capable of upholding skin, hair, nails, and body tissues [71]. Moreover, food products,including gelatin-like collagen hydrolysates, are utilized in confections, low-fat spreads, baked, andmeat products [8].

Furthermore, collagen is also popular as a food additive due to its ability to improve rheologicalproperties of meat products and act as an emulsifier in acidic products. de Castro et al. [152] reportedthat heat-treated collagen has a high potential of use as emulsifier. Heating under acidic conditionleads to reducing the charge of protein and as a consequence increasing its solubility which exerts apositive influence on the emulsion ability.

Edible films are widely used in the food industry as the barrier for moisture and oxygen toimprove the shelf life of food [71]. Food grade collagens are widely used as sausage casings and thesecasings could be developed using regenerated bovine hides [153]. Other than sausages, edible collagenfilms and coatings are also used on different meat and fish products such as hamburgers, netted roasts,boneless hams, and fish fillets [153]. The application of collagen films and coatings increases juicinessand reduces cook shrinkage in most of these foods. Moreover, the potential of using these coatingsas a protective barrier and replacing plastic wrappings has been shown to control oxidation, color,

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microbial growth, and to retain sensory attributes of meat products [71,153]. Besides, these films andcoatings could be utilized as carriers of bioactives, including antioxidants, antimicrobials, colorants,and flavorants [153].

Furthermore, collagen has the potential to be used as biobased food packaging material [154].In addition, hydrolyzed collagen may be used as a fat replacer in processed meat products like sausages.Ibrahim et al. [155] used fish collagen hydrolysates as fat replacer in the production of buffalo pattiesand reported that inclusion of hydrolyzed collagen afforded high protein, low-fat, and better texturalcharacteristics compared to the buffalo patties without hydrolyzed collagen [156].

The recent development of inclusion of collagen in beverages has gained the interest of the globalfood market. Collagen from soy, cocoa, and cappuccino, juice with collagen and birds nest drink wareexamples of some collagen-based drinks [71]. The triple helix and rod-like structure of collagen can beused for clarification of cloudy alcoholic beverages by aggregation of the yeast and other insolubleparticles [144]. Furthermore, Bilek, and Bayram [157] indicated the successful addition of hydrolyzedcollagen to beverages for enhancing their nutritional and functional properties which is now widelyused as a food ingredient in functional foods [157]. The addition of hydrolyzed collagen enhances thenutritional and functional properties of orange juice and the physicochemical and microbial propertiesof fermented dairy products [156].

However, collagen-infused liquid is generally manufactured for cosmetic purposes such asimprovement of moisture-retaining properties of the skin and prevention of forming wrinkles [157].Therefore, collagen has now gained much attention as an emerging source for cosmetic products.The cosmetic industry uses collagen as a treatment for skin replacement and other beauty-relatedproducts due to its close relationship with skin aging and abundance in the form of connective tissue inthe human body, especially in skin and bones [158]. A recent study on extraction and characterization ofcollagen from sea cucumber (Holothuria cinerascens) revealed the high moisture retention and moistureabsorption capacity compared to collagen extracted from tilapia and porcine skin [83]. Besides, PSCfrom Holothuria cinerascens were found to be rich in polar groups, including carboxyl and hydroxylgroups and capable of forming hydrogen bonds with water. This unique characteristic allows collagento interact with water hence allows its use in moisturizers [83,158].

Kim et al. [159] also studied the skin whitening and wrinkle improvement efficacy of theglycoprotein fractions from liquid extracts of boiled sea cucumber and found that glycoprotein higherthan 50 kDa fractions had the potential for use as a cosmetic ingredient. Type I collagen is themost abundant collagen type produced by skin fibroblasts. Numerous studies have proven thatcollagen derived from sea cucumbers also represents the type I collagen group. Kupper et al. [160]investigated the application of collagen/hyaluronic acid-based microemulsions from sea cucumberHolothuria cinerascens as the transdermal carrier with the focus on anti-aging research products includinganti-wrinkle creams. At this point, collagen derived from cold-water fish skin, including cod, haddock,and salmon are being widely used in the cosmetic industry [161,162].

6. Pre-Treatment, Extraction, Isolation, and Purification

Collagen exists in the insoluble macromolecular structure of the body. Therefore, procedures forthe preparation of collagen consist of several key steps including pre-treatment, extraction, separation,purification, and characterization [83,162–164]. The critical factor in collagen extraction is the removalof covalent intra- and intermolecular cross-links [162]. Collagen extraction procedure includes twosteps of pre-treatment of raw material and then extraction of collagen. Preparation procedures canvary based on the type of raw material. However, general steps, including cleaning, size reductionand pre-treatment procedures, are essential steps before extraction in order to prevent contamination.The removal of impurities may also assist in maximizing the yield and quality of the extractedcollagen [10]. Table 5 summarizes the pre-treatments and extraction methods used in collagenextraction from different sea cucumber species.

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Table 5. Pre-treatment procedures and methods used for sea cucumber collagen identification.

Sea Cucumber Species Body Parts Pre-Treatment Methods Used for Characterization ofCollagen Reference

Cucumaria frondosa Inner dermis Incubation withdeionized water

Amino acid analysisSDS-PAGE

Salt solubility determination[109]

Stichopus japonicus Body wallDisaggregation withβ-mercaptoethanol

and 0.1 M NaOH treatment

Amino acid analysisSDS-PAGE

DSC[14]

Body wall Incubation with water

Ultraviolet-visible (UV-vis) spectraSDS-PAGE

Peptide mappingAmino acid composition

DSCGel filtration chromatography

[16]

Stichopus vastus Integument Incubated with water

UV-vis spectraSDS-PAGE

peptide mappingFTIR

Gel forming capacity

[99]

Bohadshia spp. Body wall Washed in distilled water SDS-PAGE [165]

Holothuria parva Skin Washed in distilled water

SDS-PAGEDSC

Gel-forming capacityUV-vis spectra

Amino acid compositionScanning electron microscopy

[98]

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Table 5. Cont.

Sea Cucumber Species Body Parts Pre-Treatment Methods Used for Characterization ofCollagen Reference

Stichopus monotuberculatus Body wall Homogenization with water

UV-vis spectraSDS-PAGEA

mino acid analysisFTIR

Enzyme-digested peptide mappingDSC

Solubility level

[142]

Parastichopus californicus Skin and connective tissue Washed in distilled water

DSCSDS-PAGE

Enzyme-digested peptide mappingGel-forming capability

Amino acid composition

[120]

Australostichopus mollis Body wall Washed in distilled water

Scanning electron microscopyElectrophoretic analysis

Peptide mappingUV-vis spectra

DSCFTIR

Amino acid analysis

[124]

Acaudina molpadioides Body wall Soaked in 0.2 M EDTA for 48 h

Gel-filtration chromatographyAmino acid analysis

RP-HPLC and identification of peptidesequence

[127]

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6.1. Pre-Treatment

Both acidic and alkali pre-treatments are widely used in collagen extraction procedures [28].Mild chemical treatment is generally used prior to extraction, mainly due to the cross-linked nature ofcollagen [162]. For instance, acidic pre-treatment procedure is favorable for the extraction of collagenfrom raw materials with fewer cross-links, as acidic solution helps to break non-covalent bondsunder controlled temperature [57,162]. In contrast, in alkaline pre-treatment procedures, the basicsolution removes non-collagenous proteins, lipids, pigments, and calcium as well as other inorganicmaterial [10]. Factors such as time, temperature, and concentration of the solution play essential rolesfor effective removal of these non-collagenous materials during alkaline pre-treatment [161]. Accordingto Schmidt et al. [162], the concentration range of 0.05–0.10 M of NaOH can be considered as beingadequate for pre-treatment. Moreover, the same concentration range protects the acid soluble collagenand structural modifications at different temperatures from 4 to 20 ◦C. In contrast, 0.5 M NaOH causesstructural modification at 15 and 20 ◦C while 0.2 and 0.5 M both can lead to the loss of acid-solublecollagen. In addition, alkaline method is also practiced in treating thick hard raw material whichrequires effective penetration through raw material to cleave the inter- and intramolecular cross-linksof collagen (Table 4) [162,166]. Furthermore, alcohol is effective for the removal of fat and pigmentsfrom seafood and butyl alcohol is a widely used alcohol, among others [10,161].

However, most of the pre-treatments in collagen extraction from sea cucumber includeethylenediaminetetraacetic acid (EDTA) for the demineralization process [10,166]. The chelatingaction of EDTA for calcium ion facilitates the collagen extraction process by using the substrate to agreater extent [161,162].

6.2. Extraction Methods

Collagen extraction methods can be divided into two main groups as conventional and novel.According to the extraction process, both conventional and novel methods can further be classified intoseveral types such as chemical hydrolysis, enzymatic hydrolysis, ultrasound-assisted extraction, andpressurized liquid extraction (Figure 7). The yield and properties of collagen depend on the extractionmethod employed. Most of the extraction processes are carried out under controlled temperature (4 ◦C)to prevent collagen degradation [10]. Furthermore, functional properties of the extracted collagen,including the length of polypeptide chains and viscosity, solubility, water retention, emulsifying,are also affected by the extraction method. In addition, the variability of processing parameters,pre-treatment methods, storage conditions, and the nature of raw materials also influence the qualityof extracted collagen [8].

Mar. Drugs 2020, 18, x; doi: FOR PEER REVIEW www.mdpi.com/journal/marinedrugs

6.1. Pre-Treatment

Both acidic and alkali pre-treatments are widely used in collagen extraction procedures [28]. Mild chemical treatment is generally used prior to extraction, mainly due to the cross-linked nature of collagen [162]. For instance, acidic pre-treatment procedure is favorable for the extraction of collagen from raw materials with fewer cross-links, as acidic solution helps to break non-covalent bonds under controlled temperature [57,162]. In contrast, in alkaline pre-treatment procedures, the basic solution removes non-collagenous proteins, lipids, pigments, and calcium as well as other inorganic material [10]. Factors such as time, temperature, and concentration of the solution play essential roles for effective removal of these non-collagenous materials during alkaline pre-treatment [161]. According to Schmidt et al. [162], the concentration range of 0.05–0.10 M of NaOH can be considered as being adequate for pre-treatment. Moreover, the same concentration range protects the acid soluble collagen and structural modifications at different temperatures from 4 to 20 °C. In contrast, 0.5 M NaOH causes structural modification at 15 and 20 °C while 0.2 and 0.5 M both can lead to the loss of acid-soluble collagen. In addition, alkaline method is also practiced in treating thick hard raw material which requires effective penetration through raw material to cleave the inter- and intramolecular cross-links of collagen (Table 4) [162,166]. Furthermore, alcohol is effective for the removal of fat and pigments from seafood and butyl alcohol is a widely used alcohol, among others [10,161].

However, most of the pre-treatments in collagen extraction from sea cucumber include ethylenediaminetetraacetic acid (EDTA) for the demineralization process [10,166]. The chelating action of EDTA for calcium ion facilitates the collagen extraction process by using the substrate to a greater extent [161,162].

6.2. Extraction Methods

Collagen extraction methods can be divided into two main groups as conventional and novel. According to the extraction process, both conventional and novel methods can further be classified into several types such as chemical hydrolysis, enzymatic hydrolysis, ultrasound-assisted extraction, and pressurized liquid extraction (Figure 7). The yield and properties of collagen depend on the extraction method employed. Most of the extraction processes are carried out under controlled temperature (4 °C) to prevent collagen degradation [10]. Furthermore, functional properties of the extracted collagen, including the length of polypeptide chains and viscosity, solubility, water retention, emulsifying, are also affected by the extraction method. In addition, the variability of processing parameters, pre-treatment methods, storage conditions, and the nature of raw materials also influence the quality of extracted collagen [8].

Figure 7. Collagen Extraction Methods.

6.2.1. Conventional Methods

Conventional collagen extraction methods mainly include chemical hydrolysis and saltsolubilization. Acid and alkali solubilization extraction methods have been used for crude collagen

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extraction and come under the chemical hydrolysis category. The chemical hydrolysis method iswidely used over the salt solubilization method for industrial collagen production [10,162].

Salt Solubilization

When it comes to the extraction, neutral saline solutions are used due to the solubility of collagenin salt. Sodium chloride, phosphates, citrates, or Tris-HCl are mostly used neutral saline solutions [162].Collagen extraction using NaCl solution is referred to as salt-solubilized collagen. The salt solubilizationextraction method is used for collagen extraction from different tissues, including bones, cartilages,skin, and scales. However, the properties of extracted collagen are based on the salting-out method ofthe salt solubilization extraction procedure [167]. Generally, solubilization extractions are followed byeither acid or enzyme assisted extraction [168]. Ran and Wang [169] revealed the low efficiency of usingsalt solubilization for collagen extraction. Moreover, it is mandatory to control the concentration of saltdue to the nature of collagen molecules. Salt concentration < 1.0 mol L−1 is used for dissolution of type Icollagen, while concentration > 1.0 mol L−1 is best for the precipitation of type I collagen [163]. Therefore,salt or saline solution extraction has more limitations compared to chemical hydrolysis processes.

Chemical Hydrolysis

The chemical hydrolysis method is mainly categorized into acid and alkali hydrolysis. The acidhydrolysis method is extensively used and both organic and inorganic acids are able to cleave thebonds between collagen molecules and improve the extraction of collagen fibrils. Under acidicconditions, collagen molecules get more positively charged [163] and this positive charge facilitatestheir solubilization by creating the repulsion among tropocollagen molecules [10] Organic acids,including acetic, citric, lactic, and chloroacetic acid and inorganic acids such as hydrochloric acid, areused for the isolation of collagen [162,166,170]. However, organic acids are more effective compared toinorganic acids in cleaving the crosslinks of collagen molecules and result in higher extractability ofcollagen [170,171]. Acetic acid is the most commonly used organic acid which change the electrostaticnature of collagen to enhance its solubility and extractability [10,170,171].

Generally, the acid hydrolysis procedure uses 0.5 M acetic acid and the reaction mixture iscontinuously stirred for 24–72 h [169]. In order to obtain the crude collagen powder, sequentialfiltration, precipitation with NaCl and centrifugation are conducted. The filtrate should then bedissolved in acetic acid (0.5 M) followed by dialysis using 0.1 M acetic acid for two days andsubsequently distilled water for two days [162].

Some extraction requirements are varied depending on the type of raw material. For example,extraction procedure for collagen from marine sources may need to be maintained at 4 ◦C with constantstirring for 24–48 h. The resultant extraction fraction can also be varied according to the concentrationand proportion of the acid used [10,21]. de Moraes and Cunha [172] revealed that the collagen extractedunder acidic pH and high temperature possessed low molar mass and the hydrolysates formed firmergels [162]. Thus, the pH of the extraction medium may influence the nature and the physicochemicalproperties of extracted collagen. In addition, a positive relationship was reported between extractiontime and the yield of the extracted collagen [170]. However, Benjakul et al. [166] suggested that sequentialextraction cycles can give a higher yield of acid soluble collagen instead of extending the extraction time.Temperature is considered as another important variable which can directly influence the yield of collagen.Acid soluble collagen extraction can be performed within the temperature range of 4–20 ◦C withoutharming the nature of the collagen [171]. According to Pal et al. [10], acid hydrolysis process can also beconducted using 6M hydrochloric acid under high-temperature range from 110 to 120 ◦C for a longerperiod (18–48 h) with the resultant collagen being similar to that obtained under general conditions.

However, Pal et al. [10] stated that the yield of collagen could vary according to the nature ofraw material and other variables related to the extraction process. Factors including type and sourceof raw material (species, age), extraction process, concentration and proportions of acid, extractiontemperature, pH, and process time affect the yield of crude collagen. In alkali hydrolysis, strong alkali

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solutions are used for the dissolution and degradation of collagen. The two commonly used alkalisolutions are sodium and potassium hydroxide [10]. In addition, calcium oxide, calcium hydroxide,and sodium carbonate are also used as extractants [173]. Moreover, alkali has strong hydrolysis abilityand may hydrolyze proteins by acting on collagen fibrils [163,173]. However, amino acids like serine,cysteine, histidine, and threonine may be destroyed due to extreme extraction conditions [10,173].

6.2.2. Novel Methods

There are several novel methodologies for collagen extraction which address the limitationsof conventional methods. Mainly, enzymatic hydrolysis is a technique that belongs to the realm ofgreen chemistry. Furthermore, a combination of multiple methods or hybridization of chemical andenzymatic hydrolysis may be used for maximizing the yield of collagen extraction and increasing thepurity of extracted collagen. The acid-enzyme, alkali-enzyme, and acid-alkali combined hydrolysismethods have been studied for their applicability at industrial levels [163].

Different novel approaches have been investigated in collagen extraction to find the mostcost-effective procedure with minimum environmental impact. Ultrasonic [174–177] supercriticalfluid, microwave [127,178], and high-pressure extraction are under investigation in terms of industrialapplications [10]. Most of these methods need extreme conditions such as high heat and pressure.Thus, the denaturation of extracted protein might occur. However, a significant number ofstudies based on sea cucumber collagen and collagen hydrolysates have focused on enzymatichydrolysis [99,102,120,127,165].

Enzymatic Hydrolysis

Employing enzymes for the extraction of collagen is widely used and regarded as one of theconvenient biological methods for industrial application [162]. The enzymatic extraction process hasbeen developed to maximize the collagen yield as it has high reaction selectivity and less destructiveeffect on molecular structure of collagen [163]. Moreover, enzymatic hydrolysis is an efficient procedureas it possesses more favorable characteristics over the chemical hydrolysis method. Despite thehigher cost, enzymatic hydrolysis method has significant advantages compared to chemical hydrolysismethods such as high specificity, controlled degree of hydrolysis, moderate reaction conditions, finalhydrolysate with least salt content, lower waste production, and a higher yield of collagen.

Various proteolytic enzymes from animal origin (trypsin, pepsin), plant sources (bromelain, papain,ficin) or commercial proteolytic enzymes (collagenase, proteinase K, Alcalase, Nutrase, Flavourzyme,Protamex) have been used for the enzymatic hydrolysis process. Among these, pepsin from animalorigin is the most extensively used enzyme [10,166,171]. As the widely used enzyme, pepsin has theability to cleave the non-helix peptide chain of collagen protein right at the 3/4 position of N-terminal, sothe helix peptide chains of collagen remain unchanged [163]. Studies on sea cucumber collagen haveshown that pepsin solubilization extraction has no effect on its triple helix structure [14,98,99,102,142,165].Sea cucumber collagen was extracted by hydrolyzing non-helical telopeptides in cross-links usingpepsin without degenerating the integrity of the triple helix [120]. During acid hydrolysis, salt linksand Schiff base in cross-links are degenerated with weak acid. Thus, PSC has a high rate of extractioncompared to acid soluble collagen. The extraction efficiency of acid solubilized collagen (ASC) fromParastichopus californicus was lower compared to PSC [120]. Zhong et al. [101] also reported a higherPSC compared to ASC from Stichopus monotuberculatus and indicated the predominant impact ofcovalent cross-links in the telopeptide region of the peptides on collagen solubility.

Moreover, papain has also been reported to control the cleavage of the substrate protein. Jin et al. [127]used papain with microwave radiation to extract collagen from sea cucumber (Acaudina molpadioides) andreported that papain can be used to induce collagen extraction from sea cucumber body wall. However,pepsin was found as one of the best enzymes which could maintain the degree of cleavage of thesubstrate protein [162]. Pepsin soluble collagen has high purity compared to other extracted collagens,mainly due to its ability to hydrolyze non-collagenous proteins. Most of the other non-collagenous

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materials can be removed from the collagen by salt precipitation and dialysis. Besides, pepsin can alsoincrease the extraction efficiency of collagen by improving its solubility in acid solution. In addition,the degree of cross-linking at the telopeptide region of the peptides determines the yield of pepsinsoluble collagen. Adibzadeh et al. [98] reported a lower yield of pepsin soluble collagen isolated fromHolothuria parva than Parastichopus californicus and Stichopus monotuberculatus due to the higher degreeof cross-links in Holothuria parva compared to Parastichopus californicus and Stichopus monotuberculatus.

In most research efforts, exogenous enzymes are often used for the extraction process due totheir ability to control the hydrolysis with a comparatively lower processing time than other methods.However, numerous studies have been conducted to investigate the effect of endogenous enzymes ofsea cucumber species on collagen fibrils. Endogenous enzymes including cysteine proteinases [141]serine proteinases [179] and matrix metalloproteinases [97,101,118] have been characterized fromvarious sea cucumber species and are involved in the autolysis of sea cucumber. Yan et al. [179]demonstrated that serine proteinases from sea cucumber could have the ability to cleave the collagencross-links. Similar findings were revealed using trypsin-assisted (type of serine proteases) degradationof collagen fibrils isolated from Stichopus japonicus [118]. Nevertheless, cysteine and serine proteasespartially hydrolyze the surface of collagen fibrils [102,118]. Besides, metalloproteinases have alsobeen investigated to examine their activity on sea cucumber collagen. Liu et al. [119] revealed thatcollagenase type I, which belongs to the metalloproteinases, was involved in the unfolding of collagenfibrils by degenerating monomeric collagen.

Ultrasound-Assisted Extraction

Ultrasound technique is used for the extraction of collagen as an alternative to conventionalmethods in order to reduce processing time and improve the extraction yield [74]. Ultrasound is a highfrequency wave (20 kHz) which exceeds the hearing capacity of humans (16 kHz) and uses the energy ofsound waves to transfer mass by a wet process [162,180]. Energy generated by ultrasonic waves affectsthe kinetic energy of the particles in the treated substance and the phenomenon is known as sonication.Moreover, the effect of ultrasound in a liquid system or the cavitation is induced by vibration [175].The principal mechanism of ultrasound is generating bubble cavitation in the biological matrix [180].Therefore, during the process of sonication, ultrasound generates cavitation bubbles and by resultinghigh temperature and pressure, these bubbles collapse [162]. Kim et al. [175] extracted collagen fromsea bass skin using ultrasound-assisted extraction and reported no alterations in the basic structure ofthe resultant collagen. Furthermore, the yield of collagen was based on the amplitudes and duration ofthe treatment, as a higher yield was reported with higher amplitudes and short time duration. However,they recommended further studies to verify the influence of the process on structural damages to theextracted collagen. Recently, Song et al. [181] focused on developing an industrial ultrasound systemfor mass production of collagen from fish skin and reported a two-folds higher collagen yield usingultrasound-assisted extraction compared to the conventional acid-assisted extraction.

Ran and Wang [169] investigated the effect of combination of pepsin and ultrasound-assistedextraction to obtain bovine tendon derived collagen and reported a higher efficiency of extraction aswell as a better quality of extracted collagen. Li et al. [182] also indicated that the combination couldincrease the yield and reduce the required time. As a newly emerging technique, ultrasound-assistedextraction has several advantages over the conventional extraction methods, including no complexprocedures, environmentally friendly, safe to practice, short processing time, and economic viability.However, to date, no in-depth research has been conducted on identifying the effect of ultrasound onsea cucumber collagen. Thus, more research is needed for a thorough investigation on the quality ofextracted collagen as well as to overcome limitations such as controlling amplitude with the distanceand inhibition of enzyme activity [10,162,182].

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Microwave-Assisted Extraction and Other Methods

The microwave-assisted extraction process is based on the electromagnetic waves and thedisruption of the cell structure [127,183]. Microwave radiation can penetrate the interior of proteinsand facilitate the extraction by loosening their structures from the cell matrix [127]. Microwave-assistedextraction of collagen is often followed by enzyme hydrolysis as acid or enzyme-assisted hydrolysiscan be enhanced by using microwave power in order to complete the hydrolysis of collagen [183].Jin et al. [127] investigated the microwave-assisted enzymatic hydrolysis of collagen from sea cucumberAcaudina molpadioides and reported significant bioactivities of produced peptides from collagen fibrils.

Besides the microwave treatment, another recent method, high-pressure solvent extraction, wasreported for extracting collagen hydrolysates [10]. The high-pressure liquid extraction techniqueoperates at temperature and pressure within the range of 50–250 ◦C and 3.5–20 MPa, respectively [184].As high-pressure solvent exceeds its boiling temperature in most cases, water is used as an alternativeextraction solvent. Therefore, the method is usually referred to as pressurized hot water extractionor subcritical water extraction [185]. Gomez-Gullien et al. [186] investigated the extraction of gelatinfrom fish skins using high-pressure treatment and reported significantly shorter extraction time andsuperior quality gelatin compared to other conventional methods. In addition, studies on pacific bluewhiting [187], using high-pressure treatment (300–400 MPa, reported no significant effect on extractedcollagen. However, further studies are needed to confirm the quality and functionality of the resultantcollagen or collagen hydrolysates extracted using pressurized liquid extraction method.

6.3. Isolation Methods

Developing a standard isolation method for collagen becomes a difficult task mainly due tothe extreme diversity of tissues and existence of genetically distinct types of collagen. Moreover,the relationship between intermolecular interactions and collagen solubility in the solvents used areessential prior to selecting a method for isolation [93]. There are various isolation methods basedon chromatographic methods (including size exclusion, high-performance liquid, and ion-exchangechromatography, etc.), centrifugation, and solvent extraction (Figure 8).

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procedures, environmentally friendly, safe to practice, short processing time, and economic viability. However, to date, no in-depth research has been conducted on identifying the effect of ultrasound on sea cucumber collagen. Thus, more research is needed for a thorough investigation on the quality of extracted collagen as well as to overcome limitations such as controlling amplitude with the distance and inhibition of enzyme activity [10,162,182].

6.2.2.3. Microwave-Assisted Extraction and Other Methods

The microwave-assisted extraction process is based on the electromagnetic waves and the disruption of the cell structure [127,183]. Microwave radiation can penetrate the interior of proteins and facilitate the extraction by loosening their structures from the cell matrix [127]. Microwave-assisted extraction of collagen is often followed by enzyme hydrolysis as acid or enzyme-assisted hydrolysis can be enhanced by using microwave power in order to complete the hydrolysis of collagen [183]. Jin et al. [127] investigated the microwave-assisted enzymatic hydrolysis of collagen from sea cucumber Acaudina molpadioides and reported significant bioactivities of produced peptides from collagen fibrils.

Besides the microwave treatment, another recent method, high-pressure solvent extraction, was reported for extracting collagen hydrolysates [10]. The high-pressure liquid extraction technique operates at temperature and pressure within the range of 50–250 °C and 3.5–20 MPa, respectively [184]. As high-pressure solvent exceeds its boiling temperature in most cases, water is used as an alternative extraction solvent. Therefore, the method is usually referred to as pressurized hot water extraction or subcritical water extraction [185]. Gomez-Gullien et al. [186] investigated the extraction of gelatin from fish skins using high-pressure treatment and reported significantly shorter extraction time and superior quality gelatin compared to other conventional methods. In addition, studies on pacific blue whiting [187], using high-pressure treatment (300–400 MPa, reported no significant effect on extracted collagen. However, further studies are needed to confirm the quality and functionality of the resultant collagen or collagen hydrolysates extracted using pressurized liquid extraction method.

6.3. Isolation Methods

Developing a standard isolation method for collagen becomes a difficult task mainly due to the extreme diversity of tissues and existence of genetically distinct types of collagen. Moreover, the relationship between intermolecular interactions and collagen solubility in the solvents used are essential prior to selecting a method for isolation [93]. There are various isolation methods based on chromatographic methods (including size exclusion, high-performance liquid, and ion-exchange chromatography, etc.), centrifugation, and solvent extraction (Figure 8).

Figure 8. Collagen isolation methods.

6.3.1. Chromatography

Chromatography is a proven technique for separating and analyzing the components of a complexmixture, and it is most effective when the mixture is a biological extract [150,188,189]. Chromatographicseparation is always linked with the migration of the components through the column [150]. Based onprinciples such as adsorption, partition, ion-exchange, or molecular exclusion, chromatographicprocedures are used for separation purposes.

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As an effective method of protein isolation, chromatographic columns are mostly used after acentrifugation or filtration process. When considering the columns, a wide range of chromatographiccolumn packing materials are available at commercial level including gel filtration medium, ionexchange, reversed-phase packing, hydrophobic interaction adsorbent, and affinity chromatographyadsorbent [189,190].

Among the chromatographic methods, size exclusion chromatography (SEC) is extensively usedanalytical technique for quantitative and qualitative analysis of biological extracts and often usedto determine the molecular weight and molecular weight distribution [190]. Moreover, applicationof buffer exchange procedures, studies related to interaction and concentration of solutes, solutediffusivity and shape determination are considered for selecting the purification and fractionationmethods for protein aggregates [189,190].

Gel filtration is one of the widely used techniques in SEC. Cui et al. [16] carried out gel filtrationchromatography to purify the extracted collagen from Stichopus japonicus. Besides, a recent study onisolation of bioactive peptides from collagen hydrolysates from sea cucumber Acaudina molpadioidesalso used gel filtration chromatography to separate the peptides based on their molecular size [127].The authors used SEC to characterize the antioxidant peptides from microwave-assisted hydrolysatesof sea cucumber collagen and performed further analysis to determine the peptide sequences.

Numerous studies have been conducted by applying the SEC method mainly because it is mildand has minimal impact on the conformational structures of the molecules [190]. Furthermore, SEChas several favorable characteristics compared to other analytical separation methods, including itshigh recovery rate and compatibility with a range of physiological conditions. Hence, these featuresexpand the applicability of SEC in industrial level purification procedures [188–190].

Ion exchange chromatography (IEC) related to collagen identifications has been used to characterizethe crosslinks present in different collagen types. Naffa et al. [191] characterized the collagen typeI cross linked from bovine skin and used IEC as one of the isolation methods for separating thediastereoisomers of hydroxylysinonorleucine. In general, IEC is a non-denaturing technique foranalyzing and characterizing charge variants of protein samples [192]. Among the different IEC methods,cation exchange chromatography is the most efficient chromatographic method for purification andcharacterization of protein. Cation exchange columns were used to measure the collagen crosslinkspresent in tissue samples [193]. These IEC methods were employed to characterize both intact anddigest forms of proteins, including collagen [192,193].

High-Performance Liquid Chromatography (HPLC) is one of the most robust and efficientchromatographic techniques. Reversed-phase-HPLC (RP-HPLC) is used in the characterizationand purification process in collagen peptides [10]. The RP-HPLC method is often employed forthe separation of low-molecular-weight peptides and for amino acid analysis. Dong et al. [105]investigated the molecular weight distribution of collagen peptides isolated from Apostichopus japonicus.Zhu et al. [95] further purified the pepsin soluble collagen isolated from Stichopus japonicus by removingcarbohydrate moieties from the collagen fibrils and used a multi-step gradient elution system coupledwith a UV-visible spectroscopy to detect the collagen and carbohydrate peaks separately. In addition,RP-HPLC was employed to determine the amino acid sequence of the purified fractions collected fromSEC or IEC. Jin et al. [127] analyzed the fractions of collagen hydrolysates from Acaudina molpadioidesand determined the amino acid sequence of bioactive peptides separated from the SEC.

Furthermore, liquid chromatography and mass spectrometry, including matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) methods are often combined with HPLC.The primary objective of the association of sophisticated techniques with the HPLC method isto advance the identification of collagen and collagen peptides [190–193].

6.3.2. Centrifugation

Centrifugation is routinely used for the purpose of recovering precipitates, especially in the proteinpurification process. In addition, density gradient centrifugation and fractionation of subcellular

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particles and nucleic acid are the common applications of centrifugation for separation of two immiscibleliquid phases [150,162]. During centrifugation, time, velocity, and other geometrical factors related tothe rotor are dependent on the method and the type of sample [162,189,190]. In clarification procedures,refrigerated high-speed centrifugation is commonly used for any cell homogenate [150]. Centrifugationis more convenient in a laboratory scale compared to filtration. Most of the time, the standard centrifugetemperature is around 0 ◦C or below. The centrifugation step is often considered as essential in most ofthe purification methods.

6.3.3. Use of Non-Aqueous Solvents for Isolation and Purification

There are many specialized methods for protein extraction which can be used directly forchromatographic separation either after centrifugation or filtration [193,194]. Extraction yield andproperties of the resultant compound are directly linked to the extraction method [10]. The compositionof suitable extraction medium needs to be considered, including pH, buffer salts, detergents, reducingagents, proteolytic inhibitors, and bacteriostatics. Mocan et al. [194] stated that developing a standardmethod for isolation of all types of collagen from different tissues is a difficult task due to the extremediversity of both the tissue and collagen type.

6.4. Assaying of Isolated Collagen

6.4.1. Western Blotting

For collagen assay, western blot technique is used among southern and northern blot whichare generally employed for DNA and RNA assays, respectively. The western blotting technique isused to separate and identify proteins [195]. The phenomenon behind this blotting technique is totransfer electrophoretically separated macromolecules from a gel to a blotting medium. In blottingor immobilizing medium, electrophoresis pattern can be observed which allows subsequent reactionbetween separated macromolecules and probes [195,196]. In other words, through gel electrophoresis,a protein mixture is separated according to the molecular weight and type of its components, and thentransferred to the blotting medium to produce a band for each protein in order to detect the type ofprotein utilizing a specific affinity of protein [195]. Thus, three major steps are involved in the westernblotting technique to identify proteins; these include (a) separation based on molecular size; (b) transferto immobilizing medium; and (c) marking target protein using a specific or labeled antibody.

Procollagen and collagen can be identified using the western blot technique [196]. Quiñones et al. [197]used immunoblotting to study the regenerative capacity of internal organs of sea cucumberHolothuria glaberrima and western blotting results confirmed the decrease in fibrous collagen contentduring regeneration.

6.4.2. SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) can resolve theindividual components of a complex protein mixture and it is the most widely used laboratorytechnique for protein identification [188,198]. This technique is often used for fractionation andquantification of proteins in connection with either mass spectrometric identification or immunologicaltest [199]. Even though SDS-PAGE method is used as a tool to characterize proteins accordingto their size, charge, relative hydrophobicity and abundance with the newly emerging techniquessuch as protein sequencing, amino acid compositional analysis, peptide profiling hinders the use ofSDS-PAGE for analytical purposes [27,199]. Table 6 summarizes the studies conducted using differentsea cucumber species to determine the subunit composition of isolated collagen. This technique isone of the highly efficient methods of protein recovery, but there are few limitations associated with itincluding (a) relatively slow isolation rate (b) possible contamination with impurities (sodium dodecylsulphate (SDS), salts, etc.), (c) possibility of damaging the peptide chain during elution or staining andoccurrence of chemical modifications, and (d) resulting in N-terminal blockage [198,199].

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Table 6. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis methods conducted on different sea cucumber species.

Sea Cucumber Type SDS Gel Composition Collagen Type and SubunitComposition Findings Reference

Cucumaria frondosaLinear polyacrylamide gradients of4–20%, and 100 mM Tris, 3.3% SDS,

20% glycerolType I collagen (α1)3

Covalent composition of collagen is α1 trimerand amino acid composition is similar to human

collagen type I[109]

Stichopus japonicus Consisted with 9% polyacrylamide gels Type I collagen, consisting of 1 αtrimer (approximately 135 kDa)

Subunit structure of isolated collagen is similarto (α1)3 pattern that exists in the

invertebrate collagen[16]

Parastichopus californicusDiscontinuous Tris-HCl/glycine buffersystem with 7.5% resolving gel and 4%

stacking gel

Type I collagens, consisting of threeα1 chains of approximately 138 kDa

Isolated collagen constituents were α1 and βdimers and similar to that reported for collagens

from other sea cucumber species[120]

Stichopus japonicusDiscontinuous Tris-HCl/glycine buffersystem with 10% separating gel and a

5% stacking gel

Type I collagens,consisting of 1 α trimer

Electrophoresis pattern demonstrated a majorsingle band on SDS-PAGE [105]

Stichopus vastusDiscontinuous Tris-HCl-glycine buffersystem with 75 g L−1 resolving gel and

40 g L−1 stacking gel

Type I collagen, consisting of threeα1 chains of approximately

122 kDa each

Isolated collagen was consisted with majorcomponent (α1) of approximately 122 kDa and asmall amount of β dimers (about 267 kDa each)similar to that reported for collagen from other

sea cucumber species

[99]

Bohadschia spp.Discontinuous Tris-HCl-glycine buffersystem with 7.5% resolving gel and 4%

stacking gel

Type I collagen with three α1 chainswith approximately 138 kDa each

Collagen was formed with major component ofα1 and smaller amount of β dimer [165]

Stichopus monotuberculatusDiscontinuous tris-glycine buffersystem electrophoresis with 7.5%

precast gel

Type I collagen consists of three α1with molecular weight of 137 kD

Collagen consisted of 3 homologous α1 chains as(α1)3. The molecular weight of isolated collagenwas similar to the reported values of collagens

from other species

[101]

Australostichopus mollis Not included in detail Type I collagens consist of α1 andα2 chains (approximately 116 kDa)

Collagen formed α1 and α2 chains with α chainsdimer, β chains (around 212 kDa) and small

amounts of γ components and electrophoresispattern was similar to those of calf skin collagen

[124]

Holothuria cinerascens 10% SDS separating gel and 5%stacking gel

Type I collagen with identical α1chains (α1, α2 and α3)

Molecular weight of isolated α chains extractedwas about 80–90 kDa, and the molecular weight

of the β-chain was about 150–160 kDa.The reported molecular weights were

significantly lower than those of tilapia andporcine skin collagen

[83]

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6.4.3. Spectrophotometric Analysis

Purified proteins may be analyzed by ultraviolet absorbance or fluorescent spectroscopy [200].A wavelength of 200–400 nm is the most appropriate range to analyze the collagen derived from marinesources [10]. However, the maximum absorbance wavelength of collagen should include the range of210–230 nm due to the content of tyrosine, tryptophan, and phenylalanine present in collagen [16,101].

Zhu et al. [95] used UV absorbance spectroscopy at 220 and 233 nm to investigate and characterizepurified PSC content of sea cucumber Stichopus japonicus, respectively. Abedin et al. [99] used the sametechnique for characterization of collagen extracted from sea cucumber Stichopus vastus and observed asingle maximum peak at 215 nm corresponding to the UV–VIS spectrum of type I collagen.

New analytical techniques with different mass spectrometric approaches have been introducedfor protein analysis [200]. Mass spectrometry is a sensitive technique for detection, identification,and quantification of molecules based on mass to charge ratio of their ions. It provides novel meansto analyze collagen cross-links [200,201]. Samples for mass spectroscopy can come directly fromSDS-PAGE or using different protein purification methods, including chromatography. Moreover,adequate peptide solubilization before loading is one of the vital steps in sample preparation for massspectrometric analysis [201].

Fourier transform infrared (FTIR) spectroscopy is another popular technique for analyzingthe structure of proteins, especially characterization of their secondary structure. In collagencharacterization studies, FTIR spectroscopy plays a vital role as it allows confirmation by absorptionwavenumber of each amide band [105].

FTIR spectra of extracted collagens, especially from seafood by-products, indicates unique peaksof amide bands and provide evidence of the triple helical structure of collagen [10] indicating the directrelationship of amide bands and configuration of the polypeptide [202]. Generally, amide A band(3400–3440 cm−1) is related to N-H stretching vibration, amide I band is associated with stretchingvibration of the carbonyl groups along the peptide backbone while amide II is associated with the N-Hdeformation and amide III is due to C-N stretching and N-H deformation [95,99,202]. Among theamide bands, amide I band is considered as being a crucial factor in determining the secondarystructure of protein molecules. Analysis of the amide I band in infrared (IR) spectra indicates thecharacteristic structural changes of triple helix in the collagen molecule that are stabilized by hydrogenbonds present in C=O and adjacent groups [105]. Furthermore, the triple helical structure of collagenis confirmed from the absorption ratio between 1236.5 and 1449.5 cm−1 of amide III band, which isapproximately equal to 1.0 [95,99]. Lower structural stability of collagen correlates with the higherwavenumber of amide bands [105].

Abedin et al. [99] and Zhu et al. [95] used FTIR method for pepsin soluble collagen derived fromsea cucumber species and observed the absorption bands of amide I, amide II, amide III within therange 1600–1700, 1550–1600, and 1220–1320 cm−1, respectively. A recent study on thermostability of seacucumber Apostichopus japonicas used FTIR spectra to evaluate the secondary structural deformation ofcollagen during thermal treatment [105]. Thus, FTIR analysis of natural or synthetic collagen has beenwidely used to elucidate structural characteristics of collagen.

6.5. Characterization of Isolated Collagen

6.5.1. Differential Scanning Calorimetry

Thermal denaturation of collagen is a sequential and irreversible process related to the unfoldingof its unique triple helix structure [105]. The relationship between protein denaturation and thermalactivity is monitored using differential scanning calorimeter (DSC). Thermogram produced by the DSChelps in the identification of the nature of target protein under thermal stress. Midpoint or the lowestpoint of the endothermic peak in the thermogram indicates the maximum transition temperature,Tm [203], whereas Td refers to the denaturation temperature.

Mar. Drugs 2020, 18, 471 31 of 44

The helical structure of collagen is denatured and completely breaks down at 8 and 45 ◦C,respectively [202]. The thermal denaturation temperature of the collagen solution is the temperature atwhich 50% of the change in viscosity occurs. Fraction change is calculated using Equation (1).

Fraction change = [(ε2/C) − (ε3/C)]/[(ε1/C) − (ε3/C)] (1)

where C = collagen concentration (mg/mL), ε1 = specific viscosity at 8 ◦C, ε2 = specific viscosity atmeasured temperature (◦C), and ε3 = specific viscosity at 45 ◦C.

Hence, denaturation temperature is based on the changes in viscosity. The thermal determinationcurve is obtained by plotting fractional viscosities against temperature. The denaturation temperaturecan be observed where the fractional viscosity is predicted to be 0.5 [64]. Furthermore, thermaldepolymerization occurs with increasing temperature, which leads to disruption of the triple helicalstructure by breaking the hydrogen bonds [99]. The unwinding of the triple helix structure results inthe denaturation of secondary or tertiary structures of collagen, but the primary structure remainsintact. Liu et al. [103] observed similar results and depicted that thermal denaturation of sea cucumbercollagen is a time dependent-irreversible transformation of the native helical structure. The fractionalchange of PSC from the integument of sea cucumber was decreased with increasing temperature andthermal stability of collagen was correlated with the environmental and body temperature of theorganism [99]. According to most reported results, the thermostability of triple helical structure of seacucumber derived collagen is lower compared to mammalian collagen [120].

6.5.2. Tyrosine Measurement

Tyrosine content can be used for determining the collagen content of a sample [64]. Collagen maybe hydrolyzed at 105 ◦C in 6 M hydrochloric acid for 24 h under a nitrogen atmosphere and aminoacids then quantified using liquid phase ion-exchange chromatography [196]. In contrast, Lin andLiu [48] measured the tyrosine content using near UV absorption spectrum (chromophores of tyrosine).In order to analyze the purified extracted collagen, tyrosine measurement is widely used as it showsthe integrity of non-helical telopeptides and other protein contaminants [48].

6.5.3. Hydroxyproline Determination

Colorimetric assay of hydroxyproline is a robust and reliable method for analysis of collagenpurity. Collagen is rich in hydroxyproline that can be differentiated from the negligible amount presentin other proteins [204]. Moreover, hydroxyproline plays a vital role in thermal stabilization of collagenas it forms hydrogen bonds between collagen peptides. Thus, the content of hydroxyproline has adirect relationship with the thermal stability of collagen [161].

Collagen is hydrolyzed at 105 ◦C in 3.5 M sulfuric acid for 16 h to determine the hydroxyprolinecontent. The colorimetric method is performed and the hydroxyproline content is then convertedto total collagen using a factor of 7.57. The determination of collagen is usually conducted usinginternational organization for standardization (ISO) 3496: 1994 standard method for meat and meatproducts. The final value is expressed in terms of the ratio of extracted hydroxyproline compared to itsinitial concentration in the source material [161,205].

7. Functional Properties of Collagen

Interest in functional properties of collagen extracted from different sources, including animal, marineorganism, and industrial by-products, has been increasing during the past few decades. According toGomez-Guillen et al. [9], functional properties of collagen and gelatin can be divided into two maincategories as properties associated with gelling behavior, and surface behavior. Properties associatedwith gelling behavior include (a) gel formation, (b) texturing, (c) thickening, and (d) water-bindingcapacity while properties related to their surface behavior include (a) emulsification, (b) foaming andstabilization, (c) adhesion and cohesion, (d) colloid function, and (e) film formation [9,150,171].

Mar. Drugs 2020, 18, 471 32 of 44

7.1. Gelling and Hydrophilic Properties

The process of collagen gelation is the aggregation of collagen molecules that can be achieved byheating either in acid or alkali [8] and induced by alterations of processing parameters such as ionicstrength, pH, and temperature. During thermal solubilization of collagen, a considerable amount ofintra- and intermolecular cross-links are cleaved. The aqueous solution of gelatin and collagen possessesthe ability to swell by covalently linking with matrices [9]. Liu et al. [120] and Abedin et al. [99]evaluated and compared the gel-forming ability of sea cucumbers Parastichopus californicus andStichopus vastus derived collagen with calfskin collagen. The findings of these studies revealedthat ionic strength and pH were the predominant factors determining the gel-forming ability ofcollagen isolated from sea cucumbers. Moreover, calfskin collagen exhibited higher gel-forming abilitycompared to sea cucumber-derived collagen. The difference might be due to the low hydroxyprolinecontent in sea cucumber collagen which has a direct influence on creating the three-dimensionalbranched network during gel-formation [120,206].

In addition, hydrolysis may occur in some amide bonds in the primary chain of collagen moleculesduring the gelation process [8]. The gelation process of collagen, as well as gelatin, are referred asthermo reversible processes [9]. Gel strength and gel melting point are significant physical propertiesof gelatin gels [8]. The melt-in-the-mouth property of gelatin is considered as one of the significantcharacteristics of gelatin, which is extensively utilized by both food and pharmaceutical industries [8].

Hydrophilic nature and swelling ability of solubilized collagen are used to minimize the drippingloss of frozen fish and meat products [207]. Moreover, for enhancing the sensory characteristics,collagenous materials are used widely in the food industry due to their gelling properties [208].Apart from that, collagen and gelatin are utilized as wetting agents in food, pharmaceutical, andmedical applications [8,9].

Dong et al. [115] studied the changes of collagen in sea cucumber Stichopus japonicas duringcooking and reported that thermal treatments on the sea cucumber affect the appearance and thesensory properties of the final product. This is due to the alteration of water absorption ability ofcollagen. Zhu et al. [95] investigated the moisture absorption and retention capacities of PSC fromsea cucumber and suggested that PSC might be an excellent functional ingredient for cosmetics asthey exhibited a behavior comparable to that of glycerol. Li et al. [83] investigated the collagen fromsea cucumber Holothuria cinerascens and evaluated its potential application in moisturizing cosmeticproducts. They reported that the polar groups, including carboxyl (-COOH) and hydroxyl (-OH)groups on the surface of the collagen molecule, promote the moisture retention of products.

7.2. Emulsifying Properties

Charged groups of collagens contain hydrophilic or hydrophobic amino acids that are responsiblefor its surface properties. In an aqueous system, hydrophobic and hydrophilic groups are involved inreducing surface tension by moving to the surface area of the emulsion [209,210]. Hydrophobic areason the peptide chain have a major impact on the emulsifying and foaming properties of gelatin [209].

In addition, surface-active property and gel firmness are other crucial factors affecting emulsionproperties. The emulsion capacity is increased with protein concentration [9]. In addition, molecularweight also influences the stabilization of the emulsion, as high-molecular-weight gelatin forms a morestable emulsion compared to low-molecular-weight one [9,211]. Moreover, factors like temperature,pH, concentration, and homogenization of the collagen may also affect the emulsifying and foamingproperties of collagen [212]. Higher content of hydrophobic amino acid favors increased foam capacityof gelatin [211,212].

Furthermore, the stability of foams depends on various parameters including the rate of attainingequilibrium surface tension, bulk and surface viscosities, steric stabilization, and electrical repulsionbetween the two sides of the foam lamella [211].

Mar. Drugs 2020, 18, 471 33 of 44

7.3. Film Forming Properties

Biodegradable films made from edible protein-based biopolymers are gaining popularity inthe food industry due to consumers’ awareness and their low impact on the environment [213,214].However, the hygroscopic nature of gelatin limits its use as a protective barrier [9] and usuallyfollowing the extraction process, collagen molecules tend to lose their mechanical properties comparedto the native form [214]. Several investigations have been carried out to improve the mechanical andwater resistance properties of these films with the addition of other biopolymers such as chitosan,hydrophobic and hydrophilic plasticizers, lipids, and protein isolates, among others [9,214].

Avena–Bustillos et al. [215] studied the water vapor permeability of mammalian and fish gelatinfilms and found lower water permeability in fish gelatin films compared to those from mammaliansources. Moreover, water vapor permeability of cold-water and warm-water fish gelatin are alsodifferent as warm-water fish always exhibits a higher water permeability compared to that of cold-waterfish gelatin. However, excellent film-forming property of fish gelatin expands its usage in encapsulateddrugs and frozen foods. The hydrophobicity of the protein is also an essential factor for its filmformation. Notably, low hydrophobicity of marine collagen may be due to a lesser availability ofproline and hydroxyproline for hydrogen bonding with water [9].

Furthermore, the film-forming ability of collagen and collagen-based derivatives like gelatindepends on their molecular weight distribution and amino acid composition that can directly affect themechanical and barrier properties of films [216]. Recent research has been focusing on enriching thesefilms with the addition of antioxidants and antimicrobial substances to enhance their application as arenewable biomaterial [217].

8. Challenges and Future Perspectives of Sea Cucumber Collagen

Collagen as a biomaterial is now moving towards addressing certain limitations related to itsinconsistent production to meet the industrial requirement. Due to their unique characteristics,including biocompatibility and other physicochemical properties, collagens are not easily substitutedby other molecules and finding alternatives might be a difficult task. Identifying new natural sourcesof collagen and upgrading the existing methodologies for extraction, isolation, and purification can beeffective alternative solutions to overcome the existing challenges.

In this scenario, marine collagen emerges as a potential alternative source to fulfill the increasingdemand of natural collagen from other sources. Owing to its excellent biocompatibility, low riskof transmissible diseases, no or low ethical and religious constraints, marine derived collagen hasbeen recognized as a promising source of pharmaceutical and food grade commodity. Among thevarious sources of marine organisms, sea cucumber is identified as a potent, yet underexploited,source of collagen. However, collagen from marine sources contributes less than 1.5% to the totalcollagen production [9,10]. Marine animal collagens are considered as being relatively low-qualitydue to their poor rheological properties and thermal stability, mainly dictated by their amino acidcompositions which depend on the environmental and body temperature of aquatic animals. Hence, thetechno-functional feasibility of commercialization of collagen and collagen peptides derived from seacucumber may face many challenges. Existing clinical trials on the bio-efficacy of sea cucumber derivedcollagen and its derivatives are inadequate. Therefore, further exploration of functional activities ofsea cucumber derived collagen and hydrolysates thereof is urgently needed to overcome these hurdles.Moreover, it is always crucial to consider consumer acceptance, especially when incorporating collagenand its derivatives into functional food products. Usually, low-molecular-weight peptides (containamino acid residues) may impart a bitter taste to products, hence may adversely affect their sensoryattributes. Besides, the cost associated with the product is also a significant factor in gaining consumeracceptance for innovative products. Therefore, it is vital to consider consumer’s perspectives beforelaunching a new product.

In addition, issues in the commercialization process of marine-derived collagen can be resolved bydeveloping strategies to full utilization of the marine resources. Use of marine by-products (discards)

Mar. Drugs 2020, 18, 471 34 of 44

for the extraction of value-added products like collagen would be an ideal approach to maximize thesustainability and economic viability of the industry. It is also essential to consider the reproducibilityof collagen extraction along with economic viability in an industrial scale. Furthermore, beforecommercializing marine-based collagen and its derivatives, it is mandatory to consider the marketpotential, competition, overall production costs, and business environment. Therefore, efforts shouldbe directed towards exploring sea cucumber, one of the underutilized marine resources, as a potentialsource of high-value collagen peptides. Further research is needed to focus on the implementation ofnovel technologies for extraction, isolation, purification and characterization of sea cucumber derivedcollagen and their derivatives for maximizing the yield, recovery, and purity of collagen with lessimpact on the environment.

Author Contributions: Conceptualization, T.R.S., D.D., and F.S.; resources, T.R.S., D.D., and F.S.; data curation,T.R.S.; visualization, D.D., and F.S.; writing initial draft, T.R.S.; writing review and final editing, T.R.S., D.D., andF.S.; supervision, D.D., and F.S. All authors have read and agreed to the published version of the manuscript.

Funding: The research was supported by the Natural Sciences and Engineering Research Council (NSERC) ofCanada, RGPIN-2015-06121.

Conflicts of Interest: The authors declare no conflict of interest.

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